Arc-based capsids and uses thereof

ABSTRACT

Disclosed herein, in certain embodiments, are recombinant Arc and endogenous Gag polypeptides, and methods of using recombinant Arc and endogenous Gag polypeptides.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No. 17/382,092, filed Jul. 21, 2021, now U.S. Pat. No. 11,447,527, which is a Continuation of U.S. application Ser. No. 17/277,119, filed Mar. 17, 2021, which is a U.S. 371 National Stage Filing of International Application No. PCT/US2019/051786, filed Sep. 18, 2019, which claims the benefit of U.S. Provisional Patent Application No. 62/733,015, filed Sep. 18, 2018, each of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING STATEMENT

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Oct. 25, 2022, is named 54838-702-305_SL.xml and is 131,736 bytes in size.

SUMMARY OF THE DISCLOSURE

Disclosed herein, in certain embodiments, are recombinant and engineered Arc polypeptides and recombinant and engineered endogenous Gag (endo-Gag) polypeptides. In some embodiments, also included are Arc-based capsids and endo-Gag based capsids, either loaded or empty, and methods of preparing the capsids. Additionally included are methods of delivery of the Arc-based capsids and endo-Gag-based capsids to a site of interest.

Disclosed herein, in certain embodiments, is a capsid comprising a recombinant Arc polypeptide or a recombinant endogenous Gag polypeptide and a therapeutic agent. In some embodiments, the therapeutic agent is a nucleic acid. In some embodiments, the nucleic acid is an RNA. In some embodiments, the recombinant Arc polypeptide is a human Arc polypeptide comprising an amino acid sequence that is SEQ ID NO: 1 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 1. In some embodiments, the recombinant Arc polypeptide is an Arc polypeptide comprising: a) an amino acid sequence that is SEQ ID NO: 2 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 2; b) an amino acid sequence that is SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 3; c) an amino acid sequence that is SEQ ID NO: 4 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 4; d) an amino acid sequence that is SEQ ID NO: 5 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 5; e) an amino acid sequence that is SEQ ID NO: 6 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 6; f) an amino acid sequence that is SEQ ID NO: 7 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 7; g) an amino acid sequence that is SEQ ID NO: 8 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 8; h) an amino acid sequence that is SEQ ID NO: 9 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 9; i) an amino acid sequence that is SEQ ID NO: 10 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 10; or j) an amino acid sequence that is SEQ ID NO: 11 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 11; or k) an amino acid sequence that is SEQ ID NO: 12 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 12; or 1) an amino acid sequence that is SEQ ID NO: 13 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 13; or m) an amino acid sequence that is SEQ ID NO: 14 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 14; or n) an amino acid sequence that is SEQ ID NO: 15 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 15. In some embodiments, the recombinant endogenous Gag polypeptide is a human endogenous Gag polypeptide. In some embodiments, the recombinant endogenous Gag polypeptide is an endogenous Gag polypeptide comprising: a) an amino acid sequence that is SEQ ID NO: 16 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 16; b) an amino acid sequence that is SEQ ID NO: 17 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 17; c) an amino acid sequence that is SEQ ID NO: 18 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 18; d) an amino acid sequence that is SEQ ID NO: 19 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 19; e) an amino acid sequence that is SEQ ID NO: 20 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 20; f) an amino acid sequence that is SEQ ID NO: 21 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 21; or g) an amino acid sequence that is SEQ ID NO: 22 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 22; or h) an amino acid sequence that is SEQ ID NO: 23 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 23; or i) an amino acid sequence that is SEQ ID NO: 24 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 24; or j) an amino acid sequence that is SEQ ID NO: 25 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 25; or k) an amino acid sequence that is SEQ ID NO: 26 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 26; or 1) an amino acid sequence that is SEQ ID NO: 27 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 27 or m) an amino acid sequence that is SEQ ID NO: 28 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 28.

Disclosed herein, in certain embodiments, is a capsid comprising a recombinant Arc polypeptide or a recombinant endogenous Gag polypeptide, wherein the recombinant Arc polypeptide is not a rat Arc polypeptide or a human Arc polypeptide. In some embodiments, the capsid further comprises a cargo. In some embodiments, the cargo is a nucleic acid. In some embodiments, the cargo is an RNA. In some embodiments, the cargo is a therapeutic agent. In some embodiments, the recombinant Arc polypeptide is an Arc polypeptide comprising: a) an amino acid sequence that is SEQ ID NO: 2 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 2; b) an amino acid sequence that is SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 3; c) an amino acid sequence that is SEQ ID NO: 4 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 4; d) an amino acid sequence that is SEQ ID NO: 5 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 5; e) an amino acid sequence that is SEQ ID NO: 6 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 6; f) an amino acid sequence that is SEQ ID NO: 7 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 7; g) an amino acid sequence that is SEQ ID NO: 8 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 8; h) an amino acid sequence that is SEQ ID NO: 9 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 9; i) an amino acid sequence that is SEQ ID NO: 10 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 10; or j) an amino acid sequence that is SEQ ID NO: 11 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 11; or k) an amino acid sequence that is SEQ ID NO: 12 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 12; or 1) an amino acid sequence that is SEQ ID NO: 13 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 13; or m) an amino acid sequence that is SEQ ID NO: 14 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 14; or n) an amino acid sequence that is SEQ ID NO: 15 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 15. In some embodiments, the recombinant endogenous Gag polypeptide is an endogenous Gag polypeptide comprising: a) an amino acid sequence that is SEQ ID NO: 16 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 16; b) an amino acid sequence that is SEQ ID NO: 17 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 17; c) an amino acid sequence that is SEQ ID NO: 18 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 18; d) an amino acid sequence that is SEQ ID NO: 19 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 19; e) an amino acid sequence that is SEQ ID NO: 20 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 20; f) an amino acid sequence that is SEQ ID NO: 21 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 21; or g) an amino acid sequence that is SEQ ID NO: 22 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 22; or h) an amino acid sequence that is SEQ ID NO: 23 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 23; or i) an amino acid sequence that is SEQ ID NO: 24 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 24; or j) an amino acid sequence that is SEQ ID NO: 25 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 25; or k) an amino acid sequence that is SEQ ID NO: 26 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 26; or 1) an amino acid sequence that is SEQ ID NO: 27 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 27 or m) an amino acid sequence that is SEQ ID NO: 28 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 28.

Disclosed herein, in certain embodiments, is a vector comprising DNA encoding a recombinant Arc polypeptide or a recombinant endogenous Gag polypeptide. In some embodiments, the vector further encodes a therapeutic agent. In some embodiments, the therapeutic agent is a nucleic acid. In some embodiments, the nucleic acid is an RNA. In some embodiments, the recombinant Arc polypeptide is a human Arc polypeptide comprising an amino acid sequence that is SEQ ID NO: 1 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 1. In some embodiments, the recombinant Arc polypeptide is an Arc polypeptide comprising: a) an amino acid sequence that is SEQ ID NO: 2 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 2; b) an amino acid sequence that is SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 3; c) an amino acid sequence that is SEQ ID NO: 4 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 4; d) an amino acid sequence that is SEQ ID NO: 5 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 5; e) an amino acid sequence that is SEQ ID NO: 6 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 6; f) an amino acid sequence that is SEQ ID NO: 7 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 7; g) an amino acid sequence that is SEQ ID NO: 8 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 8; h) an amino acid sequence that is SEQ ID NO: 9 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 9; i) an amino acid sequence that is SEQ ID NO: 10 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 10; or j) an amino acid sequence that is SEQ ID NO: 11 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 11; or k) an amino acid sequence that is SEQ ID NO: 12 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 12; or 1) an amino acid sequence that is SEQ ID NO: 13 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 13; or m) an amino acid sequence that is SEQ ID NO: 14 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 14; or n) an amino acid sequence that is SEQ ID NO: 15 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 15. In some embodiments, the recombinant endogenous Gag polypeptide is a human endogenous Gag polypeptide. In some embodiments, the recombinant endogenous Gag polypeptide is an endogenous Gag polypeptide comprising: a) an amino acid sequence that is SEQ ID NO: 16 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 16; b) an amino acid sequence that is SEQ ID NO: 17 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 17; c) an amino acid sequence that is SEQ ID NO: 18 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 18; d) an amino acid sequence that is SEQ ID NO: 19 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 19; e) an amino acid sequence that is SEQ ID NO: 20 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 20; f) an amino acid sequence that is SEQ ID NO: 21 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 21; or g) an amino acid sequence that is SEQ ID NO: 22 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 22; or h) an amino acid sequence that is SEQ ID NO: 23 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 23; or i) an amino acid sequence that is SEQ ID NO: 24 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 24; or j) an amino acid sequence that is SEQ ID NO: 25 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 25; or k) an amino acid sequence that is SEQ ID NO: 26 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 26; or 1) an amino acid sequence that is SEQ ID NO: 27 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 27 or m) an amino acid sequence that is SEQ ID NO: 28 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 28.

Disclosed herein, in certain embodiments, is a vector comprising DNA encoding a recombinant Arc polypeptide or a recombinant endogenous Gag polypeptide, wherein the recombinant Arc polypeptide is not a rat Arc polypeptide or a human Arc polypeptide. In some embodiments, the vector further encodes a cargo. In some embodiments, the cargo is a nucleic acid. In some embodiments, the cargo is an RNA. In some embodiments, the cargo is a therapeutic agent. In some embodiments, the recombinant Arc polypeptide is an Arc polypeptide comprising: a) an amino acid sequence that is SEQ ID NO: 2 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 2; b) an amino acid sequence that is SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 3; c) an amino acid sequence that is SEQ ID NO: 4 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 4; d) an amino acid sequence that is SEQ ID NO: 5 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 5; e) an amino acid sequence that is SEQ ID NO: 6 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 6; f) an amino acid sequence that is SEQ ID NO: 7 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 7; g) an amino acid sequence that is SEQ ID NO: 8 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 8; h) an amino acid sequence that is SEQ ID NO: 9 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 9; i) an amino acid sequence that is SEQ ID NO: 10 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 10; or j) an amino acid sequence that is SEQ ID NO: 11 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 11; or k) an amino acid sequence that is SEQ ID NO: 12 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 12; or 1) an amino acid sequence that is SEQ ID NO: 13 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 13; or m) an amino acid sequence that is SEQ ID NO: 14 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 14; or n) an amino acid sequence that is SEQ ID NO: 15 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 15. In some embodiments, the recombinant endogenous Gag polypeptide is an endogenous Gag polypeptide comprising: a) an amino acid sequence that is SEQ ID NO: 16 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 16; b) an amino acid sequence that is SEQ ID NO: 17 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 17; c) an amino acid sequence that is SEQ ID NO: 18 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 18; d) an amino acid sequence that is SEQ ID NO: 19 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 19; e) an amino acid sequence that is SEQ ID NO: 20 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 20; f) an amino acid sequence that is SEQ ID NO: 21 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 21; or g) an amino acid sequence that is SEQ ID NO: 22 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 22; or h) an amino acid sequence that is SEQ ID NO: 23 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 23; or i) an amino acid sequence that is SEQ ID NO: 24 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 24; or j) an amino acid sequence that is SEQ ID NO: 25 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 25; or k) an amino acid sequence that is SEQ ID NO: 26 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 26; or 1) an amino acid sequence that is SEQ ID NO: 27 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 27 or m) an amino acid sequence that is SEQ ID NO: 28 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 28.

Disclosed herein, in certain embodiments, is a method of delivering a cargo to a cell comprising administering to the cell a capsid comprising a recombinant Arc polypeptide or a recombinant endogenous Gag polypeptide and a therapeutic agent. In some embodiments, the therapeutic agent is a nucleic acid. In some embodiments, the nucleic acid is an RNA. In some embodiments, the recombinant Arc polypeptide is a human Arc polypeptide comprising an amino acid sequence that is SEQ ID NO: 1 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 1. In some embodiments, the recombinant Arc polypeptide is an Arc polypeptide comprising: a) an amino acid sequence that is SEQ ID NO: 2 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 2; b) an amino acid sequence that is SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 3; c) an amino acid sequence that is SEQ ID NO: 4 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 4; d) an amino acid sequence that is SEQ ID NO: 5 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 5; e) an amino acid sequence that is SEQ ID NO: 6 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 6; f) an amino acid sequence that is SEQ ID NO: 7 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 7; g) an amino acid sequence that is SEQ ID NO: 8 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 8; h) an amino acid sequence that is SEQ ID NO: 9 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 9; i) an amino acid sequence that is SEQ ID NO: 10 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 10; or j) an amino acid sequence that is SEQ ID NO: 11 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 11; or k) an amino acid sequence that is SEQ ID NO: 12 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 12; or 1) an amino acid sequence that is SEQ ID NO: 13 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 13; or m) an amino acid sequence that is SEQ ID NO: 14 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 14; or n) an amino acid sequence that is SEQ ID NO: 15 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 15. In some embodiments, the recombinant endogenous Gag polypeptide is a human endogenous Gag polypeptide. In some embodiments, the recombinant endogenous Gag polypeptide is an endogenous Gag polypeptide comprising: a) an amino acid sequence that is SEQ ID NO: 16 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 16; b) an amino acid sequence that is SEQ ID NO: 17 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 17; c) an amino acid sequence that is SEQ ID NO: 18 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 18; d) an amino acid sequence that is SEQ ID NO: 19 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 19; e) an amino acid sequence that is SEQ ID NO: 20 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 20; f) an amino acid sequence that is SEQ ID NO: 21 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 21; or g) an amino acid sequence that is SEQ ID NO: 22 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 22; or h) an amino acid sequence that is SEQ ID NO: 23 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 23; or i) an amino acid sequence that is SEQ ID NO: 24 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 24; or j) an amino acid sequence that is SEQ ID NO: 25 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 25; or k) an amino acid sequence that is SEQ ID NO: 26 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 26; or 1) an amino acid sequence that is SEQ ID NO: 27 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 27 or m) an amino acid sequence that is SEQ ID NO: 28 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 28. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a vertebrate cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cargo is a nucleic acid. In some embodiments, the cell expresses a gene encoded by the nucleic acid. In some embodiments, the cargo is a therapeutic agent.

Disclosed herein, in certain embodiments, is a method of delivering a cargo to a cell comprising administering to the cell a capsid comprising a recombinant Arc polypeptide or a recombinant endogenous Gag polypeptide, wherein the recombinant Arc polypeptide is not a rat Arc polypeptide or a human Arc polypeptide. In some embodiments, the capsid further comprises a cargo. In some embodiments, the cargo is a nucleic acid. In some embodiments, the cargo is an RNA. In some embodiments, the cargo is a therapeutic agent. In some embodiments, the recombinant Arc polypeptide is an Arc polypeptide comprising: a) an amino acid sequence that is SEQ ID NO: 2 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 2; b) an amino acid sequence that is SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 3; c) an amino acid sequence that is SEQ ID NO: 4 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 4; d) an amino acid sequence that is SEQ ID NO: 5 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 5; e) an amino acid sequence that is SEQ ID NO: 6 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 6; f) an amino acid sequence that is SEQ ID NO: 7 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 7; g) an amino acid sequence that is SEQ ID NO: 8 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 8; h) an amino acid sequence that is SEQ ID NO: 9 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 9; i) an amino acid sequence that is SEQ ID NO: 10 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 10; or j) an amino acid sequence that is SEQ ID NO: 11 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 11; or k) an amino acid sequence that is SEQ ID NO: 12 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 12; or 1) an amino acid sequence that is SEQ ID NO: 13 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 13; or m) an amino acid sequence that is SEQ ID NO: 14 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 14; or n) an amino acid sequence that is SEQ ID NO: 15 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 15. In some embodiments, the recombinant endogenous Gag polypeptide is an endogenous Gag polypeptide comprising: a) an amino acid sequence that is SEQ ID NO: 16 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 16; b) an amino acid sequence that is SEQ ID NO: 17 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 17; c) an amino acid sequence that is SEQ ID NO: 18 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 18; d) an amino acid sequence that is SEQ ID NO: 19 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 19; e) an amino acid sequence that is SEQ ID NO: 20 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 20; f) an amino acid sequence that is SEQ ID NO: 21 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 21; or g) an amino acid sequence that is SEQ ID NO: 22 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 22; or h) an amino acid sequence that is SEQ ID NO: 23 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 23; or i) an amino acid sequence that is SEQ ID NO: 24 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 24; or j) an amino acid sequence that is SEQ ID NO: 25 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 25; or k) an amino acid sequence that is SEQ ID NO: 26 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 26; or 1) an amino acid sequence that is SEQ ID NO: 27 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 27 or m) an amino acid sequence that is SEQ ID NO: 28 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 28. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a vertebrate cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cargo is a nucleic acid. In some embodiments, the cell expresses a gene encoded by the nucleic acid. In some embodiments, the cargo is a therapeutic agent.

Disclosed herein, in certain embodiments, is a method of transfecting a nucleic acid into a cell comprising administering to the cell a capsid comprising a recombinant Arc polypeptide or a recombinant endogenous Gag polypeptide and a therapeutic agent. In some embodiments, the therapeutic agent is a nucleic acid. In some embodiments, the nucleic acid is an RNA. In some embodiments, the recombinant Arc polypeptide is a human Arc polypeptide comprising an amino acid sequence that is SEQ ID NO: 1 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 1. In some embodiments, the recombinant Arc polypeptide is an Arc polypeptide comprising: a) an amino acid sequence that is SEQ ID NO: 2 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 2; b) an amino acid sequence that is SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 3; c) an amino acid sequence that is SEQ ID NO: 4 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 4; d) an amino acid sequence that is SEQ ID NO: 5 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 5; e) an amino acid sequence that is SEQ ID NO: 6 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 6; f) an amino acid sequence that is SEQ ID NO: 7 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 7; g) an amino acid sequence that is SEQ ID NO: 8 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 8; h) an amino acid sequence that is SEQ ID NO: 9 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 9; i) an amino acid sequence that is SEQ ID NO: 10 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 10; or j) an amino acid sequence that is SEQ ID NO: 11 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 11; or k) an amino acid sequence that is SEQ ID NO: 12 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 12; or 1) an amino acid sequence that is SEQ ID NO: 13 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 13; or m) an amino acid sequence that is SEQ ID NO: 14 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 14; or n) an amino acid sequence that is SEQ ID NO: 15 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 15. In some embodiments, the recombinant endogenous Gag polypeptide is a human endogenous Gag polypeptide. In some embodiments, the recombinant endogenous Gag polypeptide is an endogenous Gag polypeptide comprising: a) an amino acid sequence that is SEQ ID NO: 16 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 16; b) an amino acid sequence that is SEQ ID NO: 17 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 17; c) an amino acid sequence that is SEQ ID NO: 18 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 18; d) an amino acid sequence that is SEQ ID NO: 19 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 19; e) an amino acid sequence that is SEQ ID NO: 20 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 20; f) an amino acid sequence that is SEQ ID NO: 21 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 21; or g) an amino acid sequence that is SEQ ID NO: 22 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 22; or h) an amino acid sequence that is SEQ ID NO: 23 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 23; or i) an amino acid sequence that is SEQ ID NO: 24 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 24; or j) an amino acid sequence that is SEQ ID NO: 25 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 25; or k) an amino acid sequence that is SEQ ID NO: 26 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 26; or 1) an amino acid sequence that is SEQ ID NO: 27 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 27 or m) an amino acid sequence that is SEQ ID NO: 28 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 28.

Disclosed herein, in certain embodiments, is a method of transfecting a nucleic acid into a cell comprising administering to the cell a capsid comprising a recombinant Arc polypeptide or a recombinant endogenous Gag polypeptide, wherein the recombinant Arc polypeptide is not a rat Arc polypeptide or a human Arc polypeptide. In some embodiments, the capsid further comprises a cargo. In some embodiments, the cargo is a nucleic acid. In some embodiments, the cargo is an RNA. In some embodiments, the cargo is a therapeutic agent. In some embodiments, the recombinant Arc polypeptide is an Arc polypeptide comprising: a) an amino acid sequence that is SEQ ID NO: 2 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 2; b) an amino acid sequence that is SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 3; c) an amino acid sequence that is SEQ ID NO: 4 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 4; d) an amino acid sequence that is SEQ ID NO: 5 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 5; e) an amino acid sequence that is SEQ ID NO: 6 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 6; f) an amino acid sequence that is SEQ ID NO: 7 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 7; g) an amino acid sequence that is SEQ ID NO: 8 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 8; h) an amino acid sequence that is SEQ ID NO: 9 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 9; i) an amino acid sequence that is SEQ ID NO: 10 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 10; or j) an amino acid sequence that is SEQ ID NO: 11 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 11; or k) an amino acid sequence that is SEQ ID NO: 12 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 12; or 1) an amino acid sequence that is SEQ ID NO: 13 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 13; or m) an amino acid sequence that is SEQ ID NO: 14 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 14; or n) an amino acid sequence that is SEQ ID NO: 15 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 15. In some embodiments, the recombinant endogenous Gag polypeptide is an endogenous Gag polypeptide comprising: a) an amino acid sequence that is SEQ ID NO: 12 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 12; b) an amino acid sequence that is SEQ ID NO: 13 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 13; c) an amino acid sequence that is SEQ ID NO: 14 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 14; d) an amino acid sequence that is SEQ ID NO: 15 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 15; e) an amino acid sequence that is SEQ ID NO: 16 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 16; f) an amino acid sequence that is SEQ ID NO: 17 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 17; g) an amino acid sequence that is SEQ ID NO: 18 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 18; g) an amino acid sequence that is SEQ ID NO: 19 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 19; g) an amino acid sequence that is SEQ ID NO: 20 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 20; g) an amino acid sequence that is SEQ ID NO: 21 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 21; or h) an amino acid sequence that is SEQ ID NO: 22 or an amino acid sequence that is at least 90% identical to the SEQ ID NO: 22.

Disclosed herein, in certain embodiments, is an engineered Arc or endo-Gag polypeptide comprising a cargo binding domain and at least one capsid forming subunit from an Arc or endo-Gag polypeptide. In some embodiments, the cargo binding domain comprises a nucleic acid binding domain. In some embodiments, the cargo binding domain comprises a polypeptide that binds to a small molecule. In some embodiments, the cargo binding domain comprises a polypeptide that binds to a protein, a peptide, or an antibody or binding fragment thereof. In some embodiments, the cargo binding domain comprises a polypeptide that binds to a peptidomimetic or a nucleotidomimetic. In some embodiments, the at least one capsid forming subunit comprises a polypeptide that corresponds to the CA N-lobe and/or CA C-lobe of SEQ ID NO: 1. In some embodiments, the engineered Arc or endo-Gag polypeptide further comprises a second capsid forming subunit from a different species of an Arc or endo-Gag polypeptide. In some embodiments, the second capsid forming subunit comprises a polypeptide that corresponds to the N-lobe and/or C-lobe of SEQ ID NO: 1. In some embodiments, the at least one capsid forming subunit and the second capsid forming subunit are each independently selected from a species of Arc or endo-Gag selected from a mammal, a rodent, a bird, a reptile, a fish, an insect, a fungus, or a plant. In some embodiments, the at least one capsid forming subunit and the second capsid forming subunit are from two different species. In some embodiments, the cargo binding domain is fused either directly or via a linker to the C-terminus of the at least one capsid forming subunit. In some embodiments, the cargo binding domain is fused either directly or via a linker to the N-terminus of the at least one capsid forming subunit. In some embodiments, the second capsid forming subunit is fused either directly or via a linker to the C-terminus of the at least one capsid forming subunit. In some embodiments, the second capsid forming subunit is fused either directly or via a linker to the N-terminus of the at least one capsid forming subunit. In some embodiments, the cargo binding domain is fused either directly or via a linker to the N-terminus of the at least one capsid forming subunit and the second capsid forming subunit is fused either directly or via a linker to the C-terminus of the at least one capsid forming subunit. In some embodiments, the cargo binding domain is fused either directly or via a linker to the C-terminus of the at least one capsid forming subunit and the second capsid forming subunit is fused either directly or via a linker to the N-terminus of the at least one capsid forming subunit. In some embodiments, the engineered Arc or endo-Gag polypeptide further comprises a second polypeptide. In some embodiments, the second polypeptide is fused either directly or via a linker to the at least one capsid forming subunit. In some embodiments, the second polypeptide is fused either directly or via a linker to the cargo binding domain. In some embodiments, the second polypeptide is a protein or an antibody or its binding fragments thereof. In some embodiments, the protein is a human protein or a viral protein. In some embodiments, the protein is a human Gag-like protein. In some embodiments, the protein is a de novo engineered protein designed to bind to a target receptor of interest. In some embodiments, the second polypeptide guides the delivery of a capsid formed by the engineered Arc or endo-Gag polypeptide to a target site of interest.

Disclosed herein, in certain embodiments, is a truncated Arc or endo-Gag polypeptide wherein a portion that is not involved with capsid-formation, nucleic acid binding, or delivery is removed. In some embodiments, the portion comprises a matrix (MA) domain, a reverse transcriptase (RT) domain, a nucleotide binding domain, or a combination thereof, provided that the nucleotide binding domain is not a human Arc RNA binding domain. In some embodiments, the portion comprises a CA C-lobe domain. In some embodiments, the portion comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof. In some embodiments, the N-terminal deletion comprises a deletion of up to 10 amino acids, 20 amino acids, 30 amino acids, or 50 amino acids. In some embodiments, the C-terminal deletion comprises a deletion of up to 10 amino acids, 20 amino acids, 30 amino acids, or 50 amino acids.

Disclosed herein, in certain embodiments, is an Arc or endo-Gag-based capsid comprising an engineered Arc or endo-Gag polypeptide which may be a truncated Arc or endo-Gag polypeptide and a cargo encapsulated by the capsid formed by the engineered Arc or endo-Gag polypeptide. In some embodiments, the cargo is a nucleic acid molecule. In some embodiments, the nucleic acid molecule is DNA, RNA, or a mixture of DNA and RNA. In some embodiments, the DNA and the RNA are each independently single-stranded, double-stranded, or a mixture of single and double stranded. In some embodiments, the cargo is a small molecule. In some embodiments, the cargo is a protein. In some embodiments, the cargo is a peptide. In some embodiments, the cargo is an antibody or binding fragments thereof. In some embodiments, the cargo is a peptidomimetic or a nucleotidomimetic. In some embodiments, the Arc or endo-Gag-based capsid comprises one or more additional capsid subunits from one or more species of Arc or endo-Gag proteins that are different than the engineered Arc or endo-Gag polypeptide. In some embodiments, the Arc-based or endo-Gag-based capsid comprises one or more additional capsid subunits from non-Arc proteins. In some embodiments, the one or more additional capsid subunits comprise Copia protein, ASPRV1 protein, a protein from the SCAN domain family, a protein encoded by the Paraneoplastic Ma antigen family (e.g. PNMA5, PNMA6, PNMA6A, and PNMA6B), a protein from the retrotransposon Gag-like family (e.g. RTL3, RTL6, RTL8A, RTL8B), or a combination thereof. In some embodiments, the one or more additional capsid subunits comprise BOP, LDOC1, MOAP1, PEG10, PNMA3, PNMA5, PNMA6A, PNMA6B, RTL3, RTL6, RTL8A, RTL8B, and ZNF18. In some embodiments, the capsid has a diameter of at least 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 50 nm, 80 nm, 100 nm, 120 nm, 150 nm, 200 nm, 250 nm, 300 nm, 500 nm, 600 nm, or more. In some embodiments, the capsid has a diameter of from about 1 nm to about 600 nm, from about 1 nm to about 500 nm, from about 1 nm to about 200 nm, from about 1 nm to about 100 nm, from about 1 nm to about 50 nm, or from about 1 nm to about 30 nm. In some embodiments, the capsid has a reduced off-target effect. In some embodiments, the capsid does not have an off-target effect. In some embodiments, the capsid is formed ex-vivo. In some embodiments, the capsid is formed in-vitro.

Disclosed herein, in certain embodiments, is a nucleic acid polymer encoding a recombinant or engineered Arc polypeptide or a recombinant or engineered endogenous Gag polypeptide described herein.

Disclosed herein, in certain embodiments, is a vector comprising a nucleic acid polymer encoding a recombinant or engineered Arc polypeptide or a recombinant or engineered endogenous Gag polypeptide described herein.

Disclosed herein, in certain embodiments, is a method of preparing a loaded Arc-based or endo-Gag-based capsid comprising: incubating a plurality of recombinant or engineered Arc polypeptides or a plurality of recombinant or engineered endo-Gag polypeptides with a cargo in a solution for a time sufficient to generate the loaded capsid. In some embodiments, the method further comprises mixing the solution comprising the plurality of engineered Arc or endo-Gag polypeptides with a plurality of non-Arc or non-endo-Gag capsid forming subunits prior to incubating with the cargo. In some embodiments, the plurality of non-Arc or non-endo-Gag capsid forming subunits are mixed with the plurality of recombinant or engineered Arc or endo-Gag polypeptides at a ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the plurality of non-Arc or non-endo-Gag capsid forming subunits are mixed with the plurality of engineered Arc or endo-Gag polypeptides at a ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the method further comprises mixing the solution comprising the plurality of truncated Arc or endo-Gag polypeptides with a plurality of non-Arc or endo-Gag capsid forming subunits prior to incubating with the cargo. In some embodiments, the plurality of non-Arc or endo-Gag capsid forming subunits are mixed with the plurality of truncated Arc or endo-Gag polypeptides at a ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the plurality of non-Arc or non-endo-Gag capsid forming subunits are mixed with the plurality of truncated Arc or endo-Gag polypeptides at a ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the plurality of engineered Arc or endo-Gag polypeptides is obtained from a bacterial cell system, an insect cell system, or a mammalian cell system. In some embodiments, the plurality of engineered Arc or endo-Gag polypeptides is obtained from a cell-free system. In some embodiments, the plurality of truncated Arc or endo-Gag polypeptides is obtained from a bacterial cell system, an insect cell system, or a mammalian cell system. In some embodiments, the plurality of truncated Arc or endo-Gag polypeptides is obtained from a cell-free system. In some embodiments, the loaded Arc-based or endo-Gag capsid is formulated for systemic administration. In some embodiments, the loaded Arc or endo-Gag-based capsid is formulated for local administration. In some embodiments, the loaded Arc or endo-Gag-based capsid is formulated for parenteral administration. In some embodiments, the loaded Arc or endo-Gag-based capsid is formulated for oral administration. In some embodiments, the loaded Arc or endo-Gag-based capsid is formulated for topical administration. In some embodiments, the loaded Arc or endo-Gag-based capsid is formulated for sublingual or aerosol administration.

Disclosed herein, in certain embodiments, is use of an engineered or recombinant Arc-based or endo-Gag-based capsid for delivery of a cargo to a site of interest, comprising contacting a cell at the site of interest with an Arc-based or endo-Gag-based capsid for a time sufficient to facilitate cellular uptake of the capsid. In some embodiments, the cell is a tumor cell. In some embodiments, the tumor cell is a solid tumor cell. In some embodiments, the solid tumor cell is a cell from a bladder cancer, breast cancer, brain cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, or thyroid cancer. In some embodiments, the tumor cell is from a hematologic malignancy. In some embodiments, the hematologic malignancy is a B-cell malignancy, or a T-cell malignancy. In some embodiments, the hematologic malignancy is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma, mantle cell lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, or peripheral T cell lymphoma. In some embodiments, the cell is a somatic cell. In some embodiments, the cell is a stem cell or a progenitor cell. In some embodiments, the cell is a mesenchymal stem or progenitor cell. In some embodiments, the cell is a hematopoietic stem or progenitor cell. In some embodiments, the cell is a muscle cell, a skin cell, a blood cell, or an immune cell. In some embodiments, a target protein is overexpressed or is depleted in the cell. In some embodiments, a target gene in the cell has one or more mutations. In some embodiments, the cell comprises an impaired splicing mechanism. In some embodiments, the use is an in vivo use. In some embodiments, the Arc-based capsid is administered systemically to a subject. In some embodiments, the Arc-based or endo-Gag-based capsid is administered via local administration to a subject. In some embodiments, the Arc-based or endo-Gag-based capsid is administered parenterally to a subject. In some embodiments, the Arc-based capsid is administered orally to a subject. In some embodiments, the Arc-based or endo-Gag-based capsid is administered topically to a subject. In some embodiments, the Arc-based or endo-Gag-based capsid is administered via sublingual or aerosol administration to a subject. In some embodiments, the use is an in vitro or ex vivo use.

Disclosed herein, in certain embodiments, is a kit comprising an engineered Arc or endo-Gag polypeptide, a truncated Arc or endo-Gag polypeptide, a vector encoding a recombinant or engineered Arc or endo-Gag polypeptide, or an Arc-based or endo-Gag-based capsid.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings below.

FIG. 1 is a representation of exemplary Arc polypeptides.

FIG. 2 is a representation of exemplary engineered Arc polypeptides.

FIGS. 3A and 3B illustrate an exemplary method of engineering an Arc polypeptide to carry a specific cargo (FIG. 3A) (e.g., an RNA payload), or remove an off-function effect (FIG. 3B).

FIG. 4A shows the isolation of 6×His-tagged human Arc by elution from a HisTrap column with an imidazole gradient.

FIG. 4B shows the separation of 6×His-tagged human Arc from residual nucleic acids on a mono Q column eluted with a NaCl gradient.

FIG. 5 shows a transmission electron microscope image of negatively stained human Arc capsids.

FIG. 6 shows transmission electron microscope images of negatively stained capsids formed from recombinantly expressed Arc orthologs.

FIG. 7 shows transmission electron microscope images of negatively stained capsids formed from recombinantly expressed endo-Gag proteins.

FIG. 8 shows selective internalization of Alexa594-labeled Arc capsids by HeLa cells.

FIG. 9 shows the delivery of Cre RNA to HeLa cells by Arc capsids.

FIG. 10 illustrates methods for screening Arc and endo-Gag gene candidates for the ability to transmit a heterologous RNA payload.

DETAILED DESCRIPTION OF THE DISCLOSURE

Administrating diagnostic or therapeutic agents to a site of interest with precision has presented an ongoing challenge. Available methods of delivering nucleic acids to cells have myriad limitations. For example, AAV viral vectors often used for gene therapy are immunogenic, have a limited payload capacity of <3 kb, suffer from poor bio-distribution, can only be administered by direct injection, and pose a risk of disrupting host genes by integration. Non-viral methods have different limitations. Liposomes are primarily delivered to the liver. Extracellular vesicles have a limited payload capacity of <1 kb, limited scalability, and purification difficulties. Thus, there is a recognized need for new methods of delivering therapeutic payloads.

Most molecules do not possess inherent affinity in the body. In other cases, the administered agents accumulate either in the liver and the kidney for clearance or in unintended tissue or cell types. Method for improving delivery includes coating the agent of choice with hydrophobic compounds or polymers. Such an approach increases the duration of said agent in circulation and augments hydrophobicity for cellular uptake. On the other hand, this approach does not actively direct cargo to the site of interest for delivery.

To specifically target sites where therapy is needed, therapeutic compounds are optionally fused to moieties such as ligands, antibodies, and aptamers that recognize and bind to receptors displayed on the surface of targeted cells. Upon reaching a cell of interest, the therapeutic compound is optionally further delivered to an intracellular target. For example, a therapeutic RNA can be translated to a protein if it comes into contact with a ribosome in the cytoplasm of the cell.

Arc (activity-regulated cytoskeleton-associated protein) regulates the endocytic trafficking of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) type glutamate receptors. Arc activities have been linked to synaptic strength and neuronal plasticity. Phenotypes of loss of Arc in experimental murine model included defective formation of long-term memory and reduced neuronal activity and plasticity.

Arc exhibits similar molecular properties to retroviral Gag proteins. The Arc gene may have originated from the Ty3/gypsy retrotransposon. An endogenous Gag (endo-Gag) protein is any protein endogenous to a eukaryotic organism, including Arc, that has predicted and annotated similarity to viral Gag proteins. Exemplary endo-Gag proteins are disclosed in Campillos M, Doerks T, Shah P K, and Bork P, Computational characterization of multiple Gag-like human proteins, Trends Genet. 2006 November; 22(11):585-9. An endo-Gag protein is optionally recombinantly expressed by any host cell, including a prokaryotic or eukaryotic cell, or a bacterial, yeast, insect, vertebrate, mammalian, or human cell. As described herein, in some embodiments an endo-Gag protein assembles into an endo-Gag capsid.

Disclosed herein, in certain embodiments, are Arc and endo-Gag polypeptides which assemble into a capsid for delivery of a cargo of interest. In some embodiments, also described herein are engineered Arc and endo-Gag polypeptides which assemble into a capsid for delivery of a cargo of interest. In additional embodiments, described herein are capsids, e.g., Arc-based or endo-Gag-based capsids, for delivery of a cargo of interest.

Arc Polypeptides and Endogenous Gag Polypeptides

In certain embodiments, disclosed herein is an Arc polypeptide. In certain embodiments, disclosed herein is an endo-Gag polypeptide. It should be understood that endo-Gag sequences are optional substitutes for Arc sequences to form any type of engineered Arc polypeptide described in this section.

In some instances, Arc is a non-human Arc polypeptide. In some instances, the Arc polypeptide comprises a full-length Arc polypeptide (e.g., a full-length non-human Arc polypeptide). In other instances, the Arc polypeptide comprises a fragment of non-human Arc, such as a truncated Arc polypeptide, that participates in the formation of a capsid. In additional instances, the Arc polypeptide comprises one or more domains of a non-human Arc polypeptide, in which at least one of the domains participates in the formation of a capsid. In further instances, the Arc polypeptide is a recombinant Arc polypeptide.

In some instances, endo-Gag is a non-human endo-Gag polypeptide. In some instances, the endo-Gag polypeptide comprises a full-length endo-Gag polypeptide (e.g., a full-length non-human endo-Gag polypeptide). In other instances, the endo-Gag polypeptide comprises a fragment of non-human endo-Gag, such as a truncated endo-Gag polypeptide, that participates in the formation of a capsid. In additional instances, the endo-Gag polypeptide comprises one or more domains of a non-human endo-Gag polypeptide, in which at least one of the domains participates in the formation of a capsid. In further instances, the endo-Gag polypeptide is a recombinant endo-Gag polypeptide.

In some embodiments, the Arc is a human Arc polypeptide with at least its RNA binding domain modified to bind to a cargo that is not native to the human Arc. In some instances, the Arc polypeptide comprises a full-length human Arc polypeptide with at least its RNA binding domain modified to bind to a cargo that is not native to the human Arc protein. In other instances, the Arc polypeptide comprises a human Arc fragment comprising modification(s) in at least its RNA binding domain. In additional instances, the Arc polypeptide comprises one or more domains of a human Arc polypeptide, in which at least one of the domains participates in the formation of a capsid and in which the RNA binding domain is modified to bind to a cargo that native human Arc protein does not bind to. In further instances, the Arc polypeptide is a recombinant human Arc polypeptide, with at least the RNA binding domain is modified to enable loading of a cargo that is not native to the human Arc protein.

In some embodiments, the Endo-Gag is a human Endo-Gag polypeptide with at least its RNA binding domain modified to bind to a cargo that is not native to the human endo-Gag. In some instances, the endo-Gag polypeptide comprises a full-length human endo-Gag polypeptide with at least its RNA binding domain modified to bind to a cargo that is not native to the human endo-Gag protein. In other instances, the endo-Gag polypeptide comprises a human endo-Gag fragment comprising modification(s) in at least its RNA binding domain to bind to a cargo that a native human endo-Gag protein does not bind to. In additional instances, the endo-Gag polypeptide comprises one or more domains of a human endo-Gag polypeptide, in which at least one of the domains participates in the formation of a capsid and in which the RNA binding domain is modified to bind to a cargo that is not native to the human endo-Gag protein. In further instances, the endo-Gag polypeptide is a recombinant human endo-Gag polypeptide, with at least the RNA binding domain is modified to enable loading of a cargo that is not native to the human endo-Gag protein.

In some instances, the Arc or endo-Gag polypeptide is an engineered Arc or endo-Gag polypeptide. As used herein, an engineered polypeptide is a recombinant polypeptide that is not identical in sequence to a full length, wild-type polypeptide. In some instances, the engineered Arc or endo-Gag polypeptide comprises a fragment of an Arc or endo-Gag polypeptide from a first species and at least an additional fragment from an Arc or endo-Gag polypeptide of a second species. In some cases, the first Arc or endo-Gag polypeptide is selected from a kingdom member of animalia, plantae, fungi, or protista. In some cases, the first species is selected from a mammal, a rodent, a bird, a reptile, a fish, a vertebrate, a eukaryote, an insect, a fungus, or a plant. In some cases, the second Arc polypeptide is selected from a kingdom member of animalia, plantae, fungi, or protista that is the same or different than the first Arc or endo-Gag polypeptide. In some cases, the second species is selected from a mammal, a rodent, a bird, a reptile, a fish, a vertebrate, a eukaryote, an insect, a fungus, or a plant that is different from the first species.

In some embodiments, an exemplary mammalian Arc or endo-Gag protein for expression as a recombinant or engineered Arc polypeptide is from the species Homo sapiens. Additional exemplary species of primate Arc or endo-Gag protein proteins for expression as a recombinant or engineered Arc polypeptide include: gorilla, Pongo abelii, Pan paniscus, Macaca nemestrina, Chlorocebus sabaeus, Papio anubis, Rhinopithecus roxellana, Macaca fascicularis, Nomascus leucogenys, Callithrix jacchus, Aotus nancymaae, Cebus capucinus imitator, Saimiri boliviensis boliviensis, Otolemur garnettii, Macaca mulatta, and Macaca fascicularis.

An exemplary species list of rodent Arc or endo-Gag proteins for expression as a recombinant or engineered Arc or endo-Gag polypeptide includes: Fukomys damarensis, Microcebus murinus, Heterocephalus glaber, Propithecus coquereli, Marmota marmota marmota, Galeopterus variegatus, Cavia porcellus, Dipodomys ordii, Octodon degus, Castor canadensis Nannospalax galili, Carlito syrichta, Chinchilla lanigera, Mus musculus, Ictidomys tridecemlineatus, Rattus norvegicus, Microtus ochrogaster, Otolemur garnettii, Meriones unguiculatus, Cricetulus griseus, Rattus norvegicus, Neotoma lepida, Jaculus jaculus, Mustela putorius furo, Mesocricetus auratus, Tupaia chinensis, Cricetulus griseus, Chrysochloris asiatica, Elephantulus edwardii, Erinaceus europaeus, Ochotona princeps, Sorex araneus, Monodelphis domestica, Echinops telfairi, and Condylura cristata.

An exemplary species list of Arc or endo-Gag proteins for expression as a recombinant or engineered Arc or endo-Gag polypeptide includes: Vulpes vulpes, Canis lupus dingo, Felis catus, Panthera pardus, Callorhinus ursinus, Odobenus rosmarus divergens, Equus asinus, Sus scrofa, Manis javanica, Ceratotherium simum simum, Leptonychotes weddellii, Enhydra lutris kenyoni, Lipotes vexillifer, Bos grunniens, Bubalus bubalis, Camelus dromedarius, Vicugna pacos, Orcinus orca, Neomonachus schauinslandi, Tursiops truncatus, Bos taurus, Capra hircus, Delphinapterus leucas, Ovis aries musimon, Balaenoptera acutorostrata scammoni, Neophocaena asiaeorientalis asiaeorientalis, Miniopterus natalensis, Pteropus alecto, Physeter catodon, Loxodonta africana, Orycteropus afer afer, Bos mutus, Desmodus rotundus, Hipposideros armiger, Ailuropoda melanoleuca, Trichechus manatus latirostris, Rousettus latirostris, Rousettus aegyptiacus, Eptesicus fuscus, Rhinolophus sinicus, Corvus elaphus hippelaphus, Odocoileus virginianus texanus, Pantholops hodgsonii, Camelus bactrianus, Sarcophilus harrisii, Phascolarctos cinereus, and Ornithorhynchus anatinus.

An exemplary species list of bird Arc or endo-Gag proteins for expression as a recombinant or engineered Arc or endo-Gag polypeptide includes: Gallus gallus, Corvus cornix, Cornix, Panus major, Corvus brachyrhynchos, Dromaius novaehollandiae, and Apteryx rowi.

An exemplary species list of reptile Arc protein for expression as a recombinant or engineered Arc or endo-Gag polypeptide includes: Python bivittatus, Pogona vitticeps, Anolis carolinensis, Protobothrops mucrosquamatus, Alligator sinensis, Crocodylus porosus, Gavialis gangeticus, Alligator mississippiensis, Pelodiscus sinensis, Terrapene mexicana triunguis, Chrysemys picta bellii, Chelonia mydas, Nanorana parkeri, Xenopus tropicalis, Xenopus laevis, and Latimeria chalumnae,

An exemplary species list of fish Arc protein for expression as a recombinant or engineered Arc or endo-Gag polypeptide includes: Oncorhynchus mykiss, Acanthochromis polyacanthus, Oncorhynchus kisutch, Carassius auratus, and Austrofundulus limnaeus.

An exemplary species list of insect Arc or endo-Gag proteins for expression as a recombinant or engineered Arc or endo-Gag polypeptide includes: Drosophila serrata, Drosophila bipectinata, Solenopsis invicta, Temnothorax curvispinosus, Drosophila melanogaster, Agrilus planipennis, Camponotus floridanus, Pogonomyrmex barbatus, Nilaparvata lugens, Bombyx mori, Tribolium castaneum, and Leptinotarsa decemlineata.

An exemplary species list of plant Arc or endo-Gag proteins for expression as a recombinant or engineered Arc or endo-Gag polypeptide includes Spinacia oleracea and Erythranthe guttata.

An exemplary species list of fungi proteins for expression as a recombinant or engineered Arc or endo-Gag polypeptide includes: Saccharomyces cerevisiae, Rhizopus delemar, Fusarium oxysporum, Cryptococcus neoformans, Rhizophagus irregularis, Fusarium fujikuroi, Candida albicans, Trichophyton rubrum, Pyrenophora tritici-repentis, Rhizopus microsporus, Rhizoctonia solani, Aspergillus flavus, Verticillium dahliae, Fusarium verticillioides, Aspergillus niger, Fusarium graminearum, Aspergillus fumigatus, Zymoseptoria tritici, and Trichoderma harzianum.

An exemplary species list of protists Arc or endo-Gag proteins for expression as a recombinant or engineered Arc or endo-Gag polypeptide includes: Entamoeba histolytica, Paulinella micropora, Guillardia theta, Oxyrrhis marina, Seminavis robusta, Euglena longa, Naegleria gruberi, and Trichomonas vaginalis.

In some instances, Arc or endo-Gag comprises a capsid assembly/forming (CA) domain, a cargo binding domain (e.g., an RNA binding domain), and optionally a matrix (MA) domain, a reverse transcriptase (RT) domain, or a combination thereof. In some cases, the CA domain is further divided into an N-lobe domain and a C-lobe domain. In some cases, the cargo binding domain comprises an RNA binding domain, a DNA binding domain, a protein binding domain, a peptide binding domain, an antibody binding domain, a small molecule binding domain, or a peptidomimetic/nucleotidomimetic binding domain. Exemplary cargo binding domains include, but are not limited to, domains from GPCRs, antibodies or binding fragments thereof, lipoproteins, integrins, tyrosine kinases, DNA-binding proteins, RNA-binding proteins, nucleases, ligases, proteases, integrases, isomerases, phosphatases, GTPases, aromatases, esterases, adaptor proteins, G-proteins, GEFs, cytokines, interleukins, interleukin receptors, interferons, interferon receptors, caspases, transcription factors, neurotrophic factors and their receptors, growth factors and their receptors, signal recognition particle and receptor components, extracellular matrix proteins, integral components of membrane, ribosomal proteins, translation elongation factors, translation initiation factors, GPI-anchored proteins, tissue factors, dystrophin, utrophin, dystrobrevin, any fusions, combinations, subunits, derivatives, or domains thereof.

In some embodiments, one or more non-essential regions which are not involved in capsid formation or nucleic acid binding are removed from an Arc or endo-Gag protein to generate an Arc or endo-Gag polypeptide. In such instances, one or more non-essential regions, e.g., an N-terminal region (e.g., up to 10 amino acids, up to 20 amino acids, up to 30 amino acids, or up to 50 amino acids), a C-terminal region (e.g., up to 10 amino acids, up to 20 amino acids, up to 30 amino acids, or up to 50 amino acids), a RT domain, a MA domain, or a combination thereof, are deleted from an Arc or endo-Gag protein to generate an Arc or endo-Gag polypeptide. In some cases, only the essential regions involved in capsid assembly/forming and cargo binding remain in an Arc or endo-Gag polypeptide. In additional cases, only the essential region involved in capsid assembly/forming (e.g., the N-lobe and/or the C-lobe) remains in an Arc polypeptide.

In certain embodiments, the RT domain, the MA domain, and/or the endogenous RNA binding domain are replaced with other cargo binding domains: for example, replaced with a DNA binding domain, a protein binding domain, a peptide binding domain, an antibody binding domain, a small molecule binding domain, a peptidomimetic binding domain, or a nucleotidomimetic binding domain. In some embodiments, an Arc or endo-Gag polypeptide comprises truncations or modifications of domains involved in capsid forming, nucleic acid binding, or delivery.

In some embodiments, the Arc or endo-Gag polypeptide comprises a MA domain, a CA N-lobe, a CA C-lobe, a cargo binding domain, and a RT domain. In some instances, the Arc polypeptide comprises from N-terminus to C-terminus the following domains: the MA domain, the CA N-lobe, the CA C-lobe, the RT domain, and the cargo binding domain. In some instances, the Arc or endo-Gag polypeptide comprises from N-terminus to C-terminus the following domains: the MA domain, the RT domain, the cargo binding domain, the CA N-lobe, and the CA C-lobe. In some instances, the Arc or endo-Gag polypeptide comprises from N-terminus to C-terminus the following domains: the cargo binding domain, the MA domain, the RT domain, the CA N-lobe, and the CA C-lobe. In some instances, the domains are arranged in an order that does not impede capsid assembly and cargo binding. In some instances, each of the domains is either directly or indirectly fused to the respective two flanking domains.

In some embodiments, the Arc or endo-Gag polypeptide comprises a MA domain, a CA N-lobe, a CA C-lobe, and a cargo binding domain. In some instances, the Arc or endo-Gag polypeptide comprises from N-terminus to C-terminus the following domains: the MA domain, the CA N-lobe, the CA C-lobe, and the cargo binding domain. In some instances, the Arc polypeptide comprises from N-terminus to C-terminus the following domains: the MA domain, the cargo binding domain, the CA N-lobe, and the CA C-lobe. In some instances, the Arc or endo-Gag polypeptide comprises from N-terminus to C-terminus the following domains: the cargo binding domain, the MA domain, the CA N-lobe, and the CA C-lobe. In some instances, the domains are arranged in an order that does not impede capsid assembly and cargo binding. In some instances, each of the domains is either directly or indirectly fused to the respective two flanking domains.

In some embodiments, the Arc or endo-Gag polypeptide comprises a CA N-lobe, a CA C-lobe, and a cargo binding domain. In some instances, the Arc or endo-Gag polypeptide comprises from N-terminus to C-terminus the following domains: the CA N-lobe, the CA C-lobe, and the cargo binding domain. In some instances, the Arc or endo-Gag polypeptide comprises from N-terminus to C-terminus the following domains: the cargo binding domain, the CA N-lobe, and the CA C-lobe. In some instances, the domains are arranged in an order that does not impede capsid assembly and cargo binding. In some instances, each of the domains is either directly or indirectly fused to the respective two flanking domains.

In some embodiments, the Arc or endo-Gag polypeptide comprises a CA N-lobe and a cargo binding domain. In some instances, the Arc or endo-Gag polypeptide comprises from N-terminus to C-terminus the following domains: the CA N-lobe and the cargo binding domain. In some instances, the Arc or endo-Gag polypeptide comprises from N-terminus to C-terminus the following domains: the cargo binding domain and the CA N-lobe. In some instances, the domains are arranged in an order that does not impede capsid assembly and cargo binding. In some instances, the two domains are either directly or indirectly fused to each other.

In some embodiments, the Arc or endo-Gag polypeptide is engineered to comprise a cargo binding domain, a CA domain, a MA domain, or a RT domain from one or more additional species to generate an engineered Arc polypeptide. For example, the engineered Arc or endo-Gag polypeptide comprises a cargo binding domain, a CA domain, a MA domain, or a RT domain from a first species and a cargo binding domain, a CA domain, a MA domain, or a RT domain from a second species. In some cases, the first species is selected from a eukaryote, a vertebrate, a human, a mammal, a rodent, a bird, a reptile, a fish, an insect, a fungus, or a plant. In some cases, the second species is selected from a eukaryote, a vertebrate, a human, a mammal, a rodent, a bird, a reptile, a fish, an insect, a fungus, or a plant that is different from the first species.

In some instances, the engineered or endo-Gag Arc polypeptide comprises a cargo binding domain from a first species and a CA domain (e.g., a CA N-lobe and optionally a CA C-lobe) from a second species. The engineered Arc or endo-Gag polypeptide optionally comprises a MA domain and an RT domain from either the first species or the second species. In some cases, the first species is selected from a eukaryote, a vertebrate, a human, a mammal, a rodent, a bird, a reptile, a fish, an insect, a fungus, or a plant. In some cases, the second species is selected from a eukaryote, a vertebrate, a human, a mammal, a rodent, a bird, a reptile, a fish, an insect, a fungus, or a plant that is different from the first species.

In some instances, the engineered Arc or endo-Gag polypeptide comprises a cargo binding domain, a first CA domain, a second CA domain, and optionally a MA domain and/or a RT domain. In some cases, the cargo binding domain, the first CA domain, and optionally a MA domain and/or a RT domain are from a first species and the second CA domain is from a second species. In some cases, the first CA domain is from a first species and the cargo binding domain, the second CA domain, and optionally a MA domain and/or a RT domain are from a second species. In some instances, the domains are arranged in an order that does not impede capsid assembly and cargo binding. In some instances, each of the domains is either directly or indirectly fused to the respective two adjacent domains.

In some instances, the engineered Arc or endo-Gag polypeptide comprises a cargo binding domain, a first CA domain, and a second CA domain. In some cases, the cargo binding domain and the first CA domain are from a first species and the second CA domain is from a second species. In some cases, the first CA domain is from a first species and the cargo binding domain and the second CA domain are from a second species. In such cases, the engineered Arc or endo-Gag polypeptide comprises from the N-terminus to the C-terminus the following domains: a cargo binding domain, a first CA domain, and a second CA domain. In such cases, the engineered Arc or endo-Gag polypeptide comprises from the N-terminus to the C-terminus the following domains: a first CA domain, a cargo binding domain, and a second CA domain. In such cases, the engineered Arc or endo-Gag polypeptide comprises from the N-terminus to the C-terminus the following domains: a first CA domain, a second CA domain, and a cargo binding domain. In some instances, the domains are arranged in an order that does not impede capsid assembly and cargo binding. In some instances, each of the domains is either directly or indirectly fused to the respective two flanking domains.

In some instances, the engineered Arc or endo-Gag polypeptide further comprises a second polypeptide. In some instances, the second polypeptide is fused directly or indirectly via a linker to one or more of: a cargo binding domain, a first CA domain, a second CA domain, a MA domain if present, or a RT domain if present. In some cases, the second polypeptide is a protein (e.g., a human protein), an antibody or binding fragment thereof, a viral protein, a Gag-like protein (e.g., a human Gag-like protein), or a de novo engineered protein designed to bind to a target receptor of interest. In some instances, the antibody or binding fragment thereof comprises a humanized antibody or binding fragments thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a multi-specific antibody or binding fragment thereof, a bispecific antibody or biding fragment thereof, a monovalent Fab′, a divalent Fab₂, F(ab)′3 fragments, a single-chain variable fragment (scFv), a bis-scFv, an (scFv)₂, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody or binding fragment thereof, or a chemically modified derivative thereof. In some instances, the second polypeptide guides the delivery of a capsid formed by the engineered Arc polypeptide to a target site of interest.

In some embodiments, a nucleic acid sequence or amino acid sequence of the disclosure (for example, encoding an Arc polypeptide or endo-Gag polypeptide) has at least 70% homology, at least 71% homology, at least 72% homology, at least 73% homology, at least 74% homology, at least 75% homology, at least 76% homology, at least 77% homology, at least 78% homology, at least 79% homology, at least 80% homology, at least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, at least 99% homology, at least 99.1% homology, at least 99.2% homology, at least 99.3% homology, at least 99.4% homology, at least 99.5% homology, at least 99.6% homology, at least 99.7% homology, at least 99.8% homology, at least 99.9% or at least 99.99% homology to an amino acid sequence provided herein. Various methods and software programs are used to determine the homology between two or sequences, such as NCBI BLAST, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, or another suitable method or algorithm.

In certain embodiments, the Arc polypeptide is a human polypeptide having the amino acid sequence of SEQ ID NO: 1 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1.

In certain embodiments, the Arc polypeptide is a killer whale polypeptide having the amino acid sequence of SEQ ID NO: 2 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2.

In certain embodiments, the Arc polypeptide is a white tailed deer polypeptide having the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3.

In certain embodiments, the Arc polypeptide is a platypus polypeptide having the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4.

In certain embodiments, the Arc polypeptide is a goose polypeptide having the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5.

In certain embodiments, the Arc polypeptide is a Dalmatian pelican polypeptide having the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 6.

In certain embodiments, the Arc polypeptide is a white tailed eagle polypeptide having the amino acid sequence of SEQ ID NO: 7 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7.

In certain embodiments, the Arc polypeptide is a king cobra polypeptide having the amino acid sequence of SEQ ID NO: 8 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8.

In certain embodiments, the Arc polypeptide is a ray finned fish polypeptide having the amino acid sequence of SEQ ID NO: 9 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9.

In certain embodiments, the Arc polypeptide is a sperm whale polypeptide having the amino acid sequence of SEQ ID NO: 10 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10.

In certain embodiments, the Arc polypeptide is a turkey polypeptide having the amino acid sequence of SEQ ID NO: 11 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11.

In certain embodiments, the Arc polypeptide is a central bearded dragon polypeptide having the amino acid sequence of SEQ ID NO: 12 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12.

In certain embodiments, the Arc polypeptide is a Chinese alligator polypeptide having the amino acid sequence of SEQ ID NO: 13 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13.

In certain embodiments, the Arc polypeptide is an American alligator polypeptide having the amino acid sequence of SEQ ID NO: 14 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14.

In certain embodiments, the Arc polypeptide is a Japanese gekko polypeptide having the amino acid sequence of SEQ ID NO: 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15.

In certain embodiments, the endo-Gag polypeptide is a human PNMA3 polypeptide having the amino acid sequence of SEQ ID NO: 16 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 16.

In certain embodiments, the endo-Gag polypeptide is a human PNMA5 polypeptide having the amino acid sequence of SEQ ID NO: 17 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 17.

In certain embodiments, the endo-Gag polypeptide is a human PNMA6A polypeptide having the amino acid sequence of SEQ ID NO: 18 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 18.

In certain embodiments, the endo-Gag polypeptide is a human PNMA6B polypeptide having the amino acid sequence of SEQ ID NO: 19 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 19.

In certain embodiments, the endo-Gag polypeptide is a human RTL3 polypeptide having the amino acid sequence of SEQ ID NO: 20 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 20.

In certain embodiments, the endo-Gag polypeptide is a human RTL6 polypeptide having the amino acid sequence of SEQ ID NO: 21 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 21.

In certain embodiments, the endo-Gag polypeptide is a human RTL8A polypeptide having the amino acid sequence of SEQ ID NO: 22 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 22.

In certain embodiments, the endo-Gag polypeptide is a human RTL8B polypeptide having the amino acid sequence of SEQ ID NO: 23 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 23.

In certain embodiments, the endo-Gag polypeptide is a human BOP polypeptide having the amino acid sequence of SEQ ID NO: 24 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 24.

In certain embodiments, the endo-Gag polypeptide is a human LDOC1 polypeptide having the amino acid sequence of SEQ ID NO: 25 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 25.

In certain embodiments, the endo-Gag polypeptide is a human ZNF18 polypeptide having the amino acid sequence of SEQ ID NO: 26 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26.

In certain embodiments, the endo-Gag polypeptide is a human MOAP1 polypeptide having the amino acid sequence of SEQ ID NO: 27 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 27.

In certain embodiments, the endo-Gag polypeptide is a human PEG10 polypeptide having the amino acid sequence of SEQ ID NO: 28 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 28.

In some cases, the recombinant Arc or endo-Gag polypeptide is an Arc polypeptide illustrated in FIG. 1 .

In some cases, the engineered Arc or endo-Gag polypeptide is an engineered Arc polypeptide illustrated in FIG. 2 .

Linkers

In certain embodiments, a polypeptide of the disclosure comprises a linker. In some embodiments, the linker is a peptide linker. In some instances, the linker is a rigid linker. In other instances, the linker is a flexible linker. In some cases, the linker is a non-cleavable linker. In other cases, the linker is a cleavable linker. In additional cases, the linker comprises a linear structure, or a non-linear structure (e.g., a cyclic structure).

In certain embodiments, non-cleavable linkers comprise short peptides of varying lengths. Exemplary non-cleavable linkers include (EAAAK)n (SEQ ID NO: 70), or (EAAAR)n (SEQ ID NO: 71), where n is from 1 to 5, and up to 30 residues of glutamic acid-proline or lysine-proline repeats. In some embodiments, the non-cleavable linker comprises (GGGGS)n (SEQ ID NO: 72) or (GGGS)n (SEQ ID NO: 73), wherein n is 1 to 10; KESGSVSSEQLAQFRSLD (SEQ ID NO: 74); or EGKSSGSGSESKST (SEQ ID NO: 75). In some embodiments, the non-cleavable linker comprises a poly-Gly/Ala polymer.

In certain embodiments, the linker is a cleavable linker, e.g., an extracellular cleavable linker or an intracellular cleavable linker. In some instances, the linker is designed for cleavage in the presence of particular conditions or in a particular environment (e.g., under physiological condition). For example, the design of a linker for cleavage by specific conditions, such as by a specific enzyme, allows the targeting of cellular uptake to a specific location.

In some embodiments, the linker is a pH-sensitive linker. In one instance, the linker is cleaved under basic pH conditions. In other instance, the linker is cleaved under acidic pH conditions.

In some embodiments, the linker is cleaved in vivo by endogenous enzymes (e.g., proteases) such as serine proteases including but not limited to thrombin, metalloproteases, furin, cathepsin B, necrotic enzymes (e.g., calpains), and the like. Exemplary cleavable linkers include, but are not limited to, GGAANLVRGG (SEQ ID NO: 76); SGRIGFLRTA (SEQ ID NO: 77); SGRSA (SEQ ID NO: 78); GFLG (SEQ ID NO: 79); ALAL (SEQ ID NO: 80); FK; PIC(Et)F-F (SEQ ID NO: 81), where C(Et) indicates S-ethylcysteine; PR(S/T)(L/I)(S/T) (SEQ ID NO: 82); DEVD (SEQ ID NO: 83); GWEHDG (SEQ ID NO: 84); RPLALWRS (SEQ ID NO: 85); or a combination thereof.

Capsids

In some embodiments, disclosed herein is a capsid. In some instances, the capsid comprises an Arc polypeptide and/or an endo-Gag polypeptide such as a Copia protein, ASPRV1 protein, a protein from the SCAN domain family, a protein encoded by the Paraneoplastic Ma antigen family, a protein or a combination of proteins chosen from the retrotransposon Gag-like family, or a combination thereof. Exemplary endo-Gag polypeptides are BOP, LDOC1, MOAP1, PEG10, PNMA3, PNMA5, PNMA6A, PNMA6B, RTL3, RTL6, RTL8A, RTL8B, and ZNF18.

In some instances, the Arc polypeptide, the Copia protein, the ASPRV1 protein, the protein from the SCAN domain family, the protein encoded by the Paraneoplastic Ma antigen family, and the protein or a combination of proteins chosen from the retrotransposon Gag-like family are each independently a full-length polypeptide. In other instances, the Arc polypeptide, the Copia protein, the ASPRV1 protein, the protein from the SCAN domain family, the protein encoded by the Paraneoplastic Ma antigen family, and the protein or a combination of proteins chosen from the retrotransposon Gag-like family are each independently a functional fragment thereof, e.g., that is capable of forming a subunit of a capsid.

Arc-Based Capsids and Endo-Gag-Based Capsids

In some embodiments, the capsid comprises an Arc-based capsid. In some embodiments, the capsid comprises an endo-Gag-based capsid. In some instances, the Arc-based and/or endo-Gag capsid comprises a plurality of recombinant Arc polypeptides and/or endo-Gag polypeptides described above, a plurality of engineered Arc polypeptides and/or endo-Gag polypeptides described above, or a combination thereof. In some cases, the Arc-based capsid comprises a plurality of recombinant Arc polypeptides. In other cases, the Arc-based capsid comprises a plurality of engineered Arc polypeptides. In some cases, the endo-Gag-based capsid comprises a plurality of recombinant endo-Gag polypeptides. In other cases, the endo-Gag-based capsid comprises a plurality of engineered endo-Gag polypeptides.

In some embodiments, the Arc-based or endo-Gag-based capsid comprises a first plurality of Arc and/or endo-Gag polypeptides from a first species and a second plurality of Arc and/or endo-Gag polypeptides from at least a second species. In some cases, the first species is selected from a eukaryote, a vertebrate, a human, a mammal, a rodent, a bird, a reptile, a fish, an insect, a fungus, or a plant. In some cases, the second species is selected from a eukaryote, a vertebrate, a human, a mammal, a rodent, a bird, a reptile, a fish, an insect, a fungus, or a plant that is different from the first species.

In some instances, the ratio of the first plurality of Arc or endo-Gag polypeptides to the second plurality of Arc or endo-Gag polypeptides is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, or 100:1. In some cases, the ratio of the first plurality of Arc or endo-Gag polypeptides to the second plurality of Arc or endo-Gag polypeptides is 1:1. In some cases, the ratio of the first plurality of Arc or endo-Gag polypeptides to the second plurality of Arc or endo-Gag polypeptides is 2:1. In some cases, the ratio of the first plurality of Arc or endo-Gag polypeptides to the second plurality of Arc or endo-Gag polypeptides is 4:1. In some cases, the ratio of the first plurality of Arc or endo-Gag polypeptides to the second plurality of Arc or endo-Gag polypeptides is 5:1. In some cases, the ratio of the first plurality of Arc or endo-Gag polypeptides to the second plurality of Arc or endo-Gag polypeptides is 8:1. In some cases, the ratio of the first plurality of Arc or endo-Gag polypeptides to the second plurality of Arc polypeptides is 10:1. In some cases, the ratio of the first plurality of Arc or endo-Gag polypeptides to the second plurality of Arc or endo-Gag polypeptides is 20:1. In some cases, the ratio of the first plurality of Arc or endo-Gag polypeptides to the second plurality of Arc or endo-Gag polypeptides is 50:1. In some cases, the ratio of the first plurality of Arc or endo-Gag polypeptides to the second plurality of Arc or endo-Gag polypeptides is 100:1. In some instances, the ratio is the comparison in molar concentration. In some instances, the ratio is the comparison in the number of capsid forming subunits (e.g., each of the or engineered Arc polypeptide forms a capsid subunit).

In some instances, the ratio of the first plurality of Arc or endo-Gag polypeptides to the second plurality of Arc or endo-Gag polypeptides is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, or 1:50. In some cases, the ratio of the first plurality of Arc or endo-Gag polypeptides to the second plurality of Arc or endo-Gag polypeptides is 1:2. In some cases, the ratio of the first plurality of Arc or endo-Gag polypeptides to the second plurality of Arc or endo-Gag polypeptides is 1:5. In some cases, the ratio of the first plurality of Arc or endo-Gag polypeptides to the second plurality of Arc or endo-Gag polypeptides is 1:8. In some cases, the ratio of the first plurality of Arc or endo-Gag polypeptides to the second plurality of Arc or endo-Gag polypeptides is 1:10. In some cases, the ratio of the first plurality of Arc or endo-Gag polypeptides to the second plurality of Arc or endo-Gag polypeptides is 1:20. In some cases, the ratio of the first plurality of Arc or endo-Gag polypeptides to the second plurality of Arc or endo-Gag polypeptides is 1:50. In some instances, the ratio is the comparison in molar concentration. In some instances, the ratio is the comparison in the number of capsid forming subunits (e.g., each of the recombinant or engineered Arc or endo-Gag polypeptide forms a capsid subunit).

In some embodiments, the Arc-based capsid or endo-Gag-based capsid comprises a plurality of recombinant or engineered Arc polypeptides and a plurality of non-Arc proteins. Exemplary species of non-Arc proteins include but are not limited to, Copia, ASPRV1, a protein or a combination of proteins chosen from the SCAN domain family, a protein or a combination of proteins chosen from the Paraneoplastic Ma antigen family, and a protein or a combination of proteins chosen from the retrotransposon Gag-like family. Exemplary species of non-Arc proteins include BOP, LDOC1, MOAP1, PEG10, PNMA3, PNMA5, PNMA6A, PNMA6B, RTL3, RTL6, RTL8A, RTL8B, and ZNF18.

In some instances, the ratio of the plurality of recombinant or engineered Arc polypeptides to the plurality of non-Arc proteins is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, or 100:1. In some cases, the ratio of the plurality of recombinant or engineered Arc polypeptides to the plurality of non-Arc proteins is 1:1. In some cases, the ratio of the plurality of recombinant or engineered Arc polypeptides to the plurality of non-Arc proteins is 2:1. In some cases, the ratio of the plurality of recombinant or engineered Arc polypeptides to the plurality of non-Arc proteins is 4:1. In some cases, the ratio of the plurality of recombinant or engineered Arc polypeptides to the plurality of non-Arc proteins is 5:1. In some cases, the ratio of the plurality of recombinant or engineered Arc polypeptides to the plurality of non-Arc proteins is 8:1. In some cases, the ratio of the plurality of recombinant or engineered Arc polypeptides to the plurality of non-Arc proteins is 10:1. In some cases, the ratio of the plurality of recombinant or engineered Arc polypeptides to the plurality of non-Arc proteins is 20:1. In some cases, the ratio of the plurality of recombinant or engineered Arc polypeptides to the plurality of non-Arc proteins is 50:1. In some cases, the ratio of the plurality of recombinant or engineered Arc polypeptides to the plurality of non-Arc proteins is 100:1. In some instances, the ratio is the comparison in molar concentration. In some instances, the ratio is the comparison in the number of capsid forming subunits (e.g., each of the recombinant or engineered Arc polypeptide forms a capsid subunit).

In some instances, the ratio of the plurality of recombinant or engineered Arc polypeptides to the plurality of non-Arc proteins is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, or 1:50. In some cases, the ratio of the plurality of recombinant or engineered Arc polypeptides to the plurality of non-Arc proteins is 1:2. In some cases, the ratio of the plurality of recombinant or engineered Arc polypeptides to the plurality of non-Arc proteins is 1:5. In some cases, the ratio of the plurality of recombinant or engineered Arc polypeptides to the plurality of non-Arc proteins is 1:8. In some cases, the ratio of the plurality of recombinant or engineered Arc polypeptides to the plurality of non-Arc proteins is 1:10. In some cases, the ratio of the plurality of recombinant or engineered Arc polypeptides to the plurality of non-Arc proteins is 1:20. In some cases, the ratio of the plurality of recombinant or engineered Arc polypeptides to the plurality of non-Arc proteins is 1:50. In some instances, the ratio is the comparison in molar concentration. In some instances, the ratio is the comparison in the number of capsid forming subunits (e.g., each of the recombinant or engineered Arc polypeptide forms a capsid subunit).

In some embodiments, the capsid has a diameter of at least 1 nm, or more. In some instances, the capsid has a diameter of at least 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, or more. In some instances, the capsid has a diameter of at least 5 nm, or more. In some cases, the capsid has a diameter of at least 10 nm, or more. In some instances, the capsid has a diameter of at least 20 nm, or more. In some cases, the capsid has a diameter of at least 30 nm, or more. In some cases, the capsid has a diameter of at least 40 nm, or more. In some cases, the capsid has a diameter of at least 50 nm, or more. In some cases, the capsid has a diameter of at least 80 nm, or more. In some cases, the capsid has a diameter of at least 100 nm, or more. In some cases, the capsid has a diameter of at least 200 nm, or more. In some cases, the capsid has a diameter of at least 300 nm, or more. In some cases, the capsid has a diameter of at least 400 nm, or more. In some cases, the capsid has a diameter of at least 500 nm, or more. In some cases, the capsid has a diameter of at least 600 nm, or more.

In some embodiments, the capsid has a diameter of at most 1 nm, or less. In some instances, the capsid has a diameter of at most 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, or less. In some instances, the capsid has a diameter of at most 5 nm, or less. In some cases, the capsid has a diameter of at most 10 nm, or less. In some instances, the capsid has a diameter of at most 20 nm, or less. In some cases, the capsid has a diameter of at most 30 nm, or less. In some cases, the capsid has a diameter of at least 40 nm, or less. In some cases, the capsid has a diameter of at least 50 nm, or less. In some cases, the capsid has a diameter of at least 80 nm, or less. In some cases, the capsid has a diameter of at least 100 nm, or less. In some cases, the capsid has a diameter of at least 200 nm, or less. In some cases, the capsid has a diameter of at least 300 nm, or less. In some cases, the capsid has a diameter of at least 400 nm, or less. In some cases, the capsid has a diameter of at least 500 nm, or less. In some cases, the capsid has a diameter of at least 600 nm, or less.

In some embodiments, the capsid has a diameter of about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 300 nm, 400 nm, 500 nm, or 600 nm. In some instances, the capsid has a diameter of about 5 nm. In some cases, the capsid has a diameter of about 10 nm. In some instances, the capsid has a diameter of about 20 nm. In some cases, the capsid has a diameter of about 30 nm. In some cases, the capsid has a diameter of about 40 nm. In some cases, the capsid has a diameter of about 50 nm. In some cases, the capsid has a diameter of about 80 nm. In some cases, the capsid has a diameter of about 100 nm. In some cases, the capsid has a diameter of about 200 nm. In some cases, the capsid has a diameter of about 300 nm. In some cases, the capsid has a diameter of about 400 nm. In some cases, the capsid has a diameter of about 500 nm. In some cases, the capsid has a diameter of about 600 nm.

In some embodiments, the capsid has a diameter of from about 1 nm to about 600 nm. In some instances, the capsid has a diameter of from about 2 nm to about 500 nm, from about 2 nm to about 400 nm, from about 2 nm to about 300 nm, from about 2 nm to about 200 nm, from about 2 nm to about 100 nm, from about 2 nm to about 50 nm, from about 2 nm to about 30 nm, from about 20 nm to about 400 nm, from about 20 nm to about 300 nm, from about 20 nm to about 200 nm, from about 20 nm to about 100 nm, from about 20 nm to about 50 nm, from about 20 nm to about 30 nm, from about 30 nm to about 500 nm, from about 30 nm to about 400 nm, from about 30 nm to about 300 nm, from about 30 nm to about 200 nm, from about 30 nm to about 100 nm, from about 30 nm to about 50 nm, from about 50 nm to about 300 nm, from about 50 nm to about 200 nm, from about 50 nm to about 100 nm, from about 2 nm to about 25 nm, from about 2 nm to about 20 nm, from about 2 nm to about 10 nm, from about 5 nm to about 25 nm, from about 5 nm to about 20 nm, from about 5 nm to about 10 nm, from about 10 nm to about 25 nm, or from about 10 nm to about 20 nm.

In some embodiments, the capsid has a reduced off-target effect. In some cases, the off-target effect is less than 10%, 5%, 4%, 3%, 2%, 1%, or 0.5%. In some cases, the off-target effect is no more than 10%, 5%, 4%, 3%, 2%, 1%, or 0.5%.

In some cases, the capsid does not have an off-target effect.

In certain embodiments, the formation of Arc and/or endo-Gag-based capsids occurs either ex vivo or in vitro.

In some instances, the Arc and/or endo-Gag-based capsids is assembled in vivo.

In some instances, the Arc and/or endo-Gag-based capsids is stable at room temperature. In some cases, the Arc and/or endo-Gag-based capsids is empty. In other cases, the Arc and/or endo-Gag-based capsids is loaded (for example, loaded with a cargo and/or a therapeutic agent, e.g., a DNA or an RNA).

In some instances, the Arc and/or endo-Gag-based capsids is stable at a temperature from about 2° C. to about 37° C. In some instances, the Arc and/or endo-Gag-based capsids is stable at a temperature from about 2° C. to about 8° C., about 2° C. to about 4° C., about 20° C. to about 37° C., about 25° C. to about 37° C., about 20° C. to about 30° C., about 25° C. to about 30° C., or about 30° C. to about 37° C. In some cases, the Arc and/or endo-Gag-based capsid is empty. In other cases, the Arc and/or endo-Gag-based capsids is loaded (for example, loaded with a cargo and/or a therapeutic agent, e.g., a DNA or an RNA).

In some instances, the Arc and/or endo-Gag-based capsids is stable for at least about 1 day, 2 days, 4 days, 5 days, 7 days, 14 days, 28 days, 30 days, 60 days, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 18 months, 24 months, 3 years, 5 years, or longer. In some case, the Arc and/or endo-Gag-based capsids has minimum degradation, e.g., less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5% based on the total population of the Arc and/or endo-Gag-based capsids that is degraded. In some cases, the Arc and/or endo-Gag-based capsid is empty. In other cases, the Arc and/or endo-Gag-based capsids is loaded (for example, loaded with a therapeutic agent, e.g., a DNA or an RNA).

Additional Capsids

In some embodiments, the capsid comprises the Copia protein. In some instances, the Copia protein is from Drosophila melanogaster (UniProtKB—P04146), Ceratitis capitate (UniProtKB—W8BHY5), or Drosophila simulans (UniProtKB—Q08461).

In some embodiments, the capsid comprises the protein ASPRV1. The ASPRV1 protein is a structural protein that participates in the development and maintenance of the skin barrier. In some instances, the protein ASPRV1 is from Homo sapiens (UniProtKB— Q53RT3).

In some embodiments, the capsid comprises a protein from the SCAN domain family. SCAN domain is a superfamily of zinc finger transcription factors. SCAN domain is also known as leucine rich region (LeR) and functions as protein interaction domain that mediates self-association or selective association with other proteins.

In some embodiments, the capsid comprises a protein from the Paraneoplastic Ma antigen family. The Paraneoplastic Ma antigen family comprises about 14 members of neuro- and testis-specific proteins.

In some embodiments, the capsid comprises a protein encoded by a Retrotransposon Gag-like gene.

In some embodiments, the capsid comprises BOP, LDOC1, MOAP1, PEG10, PNMA3, PNMA5, PNMA6A, PNMA6B, RTL3, RTL6, RTL8A, RTL8B, and/or ZNF18.

Cargos

In some embodiments, a composition of the disclosure (for example, a capsid) comprises a cargo. In some embodiments, the cargo is a therapeutic agent. In some embodiments, the cargo is a nucleic acid molecule, a small molecule, a protein, a peptide, an antibody or binding fragment thereof, a peptidomimetic, or a nucleotidomimetic. In some instances, the cargo is a therapeutic cargo, comprising e.g., one or more drugs. In some instances, the cargo comprises a diagnostic tool, for profiling, e.g., one or more markers (such as markers associates with one or more disease phenotypes). In additional instances, the cargo comprises an imaging tool.

In some instances, the cargo is a nucleic acid molecule. Exemplary nucleic acid molecules include DNA, RNA, or a mixture of DNA and RNA. In some instances, the nucleic acid molecule is a DNA polymer. In some cases, the DNA is a single stranded DNA polymer. In other cases, the DNA is a double stranded DNA polymer. In additional cases, the DNA is a hybrid of single and double stranded DNA polymer.

In some embodiments, the nucleic acid molecule is a RNA polymer, e.g., a single stranded RNA polymer, a double stranded RNA polymer, or a hybrid of single and double stranded RNA polymers. In some instances, the RNA comprises and/or encodes an antisense oligoribonucleotide, a siRNA, an mRNA, a tRNA, an rRNA, a snRNA, a shRNA, microRNA, or a non-coding RNA.

In some embodiments, the nucleic acid molecule comprises a hybrid of DNA and RNA.

In some embodiments, the nucleic acid molecule is an antisense oligonucleotide, optionally comprising DNA, RNA, or a hybrid of DNA and RNA.

In some instances, the nucleic acid molecule comprises and/or encodes an mRNA molecule.

In some embodiments, the nucleic acid molecule comprises and/or encodes an RNAi molecule. In some cases, the RNAi molecule is a microRNA (miRNA) molecule. In other cases, the RNAi molecule is a siRNA molecule. The miRNA and/or siRNA are optionally double-stranded or as a hairpin, and further optionally encapsulated as precursor molecules.

In some embodiments, the nucleic acid molecule is for use in a nucleic acid-based therapy. In some instances, the nucleic acid molecule is for regulating gene expression (e.g., modulating mRNA translation or degradation), modulating RNA splicing, or RNA interference. In some cases, the nucleic acid molecule comprises and/or encodes an antisense oligonucleotide, microRNA molecule, siRNA molecule, mRNA molecule, for use in regulation of gene expression, modulating RNA splicing, or RNA interference.

In some instances, the nucleic acid molecule is for use in gene editing. Exemplary gene editing systems include, but are not limited to, CRISPR-Cas systems, zinc finger nuclease (ZFN) systems, and transcription activator-like effector nuclease (TALEN) systems. In some cases, the nucleic acid molecule comprises and/or encodes a component involved in the CRISPR-Cas systems, ZFN systems, or the TALEN systems.

In some cases, the nucleic acid molecule is for use in antigen production for therapeutic and/or prophylactic vaccine production. For example, the nucleic acid molecule encodes an antigen that is expressed and elicits a desirable immune response (e.g., a pro-inflammatory immune response, an anti-inflammatory immune response, an B cell response, an antibody response, a T cell response, a CD4+ T cell response, a CD8+ T cell response, a Th1 immune response, a Th2 immune response, a Th17 immune response, a Treg immune response, or a combination thereof).

In some cases, the nucleic acid molecule comprises a nucleic acid enzyme. Nucleic acid enzymes are RNA molecules (e.g., ribozymes) or DNA molecules (e.g., deoxyribozymes) that have catalytic activities. In some instances, the nucleic acid molecule is a ribozyme. In other instances, the nucleic acid molecule is a deoxyribozyme. In some cases, the nucleic acid molecule is a MNAzyme, which functions as a biosensor and/or a molecular switch (see, e.g., Mokany, et al., “MNAzymes, a versatile new class of nucleic acid enzymes that can function as biosensors and molecular switches,” JACS 132(2): 1051-1059 (2010)).

In some instances, exemplary targets of the nucleic acid molecule include, but are not limited to, UL123 (human cytomegalovirus), APOB, AR (androgen receptor) gene, KRAS, PCSK9, CFTR, and SMN (e.g., SMN2).

In some embodiments, the nucleic acid molecule is at least 5 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 10 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 12 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 15 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 18 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 19 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 20 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 21 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 22 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 23 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 24 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 25 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 26 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 27 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 28 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 29 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 30 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 40 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 50 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 100 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 200 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 300 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 500 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 1000 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 2000 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 3000 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 4000 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 5000 nucleotides or more in length. In some instances, the nucleic acid molecule is at least 8000 nucleotides or more in length.

In some embodiments, the nucleic acid molecule is at most 12 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 15 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 18 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 19 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 20 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 21 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 22 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 23 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 24 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 25 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 26 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 27 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 28 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 29 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 30 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 40 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 50 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 100 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 200 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 300 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 500 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 1000 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 2000 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 3000 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 4000 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 5000 nucleotides or less in length. In some instances, the nucleic acid molecule is at most 8000 nucleotides or less in length.

In some embodiments, the nucleic acid molecule is about 5 nucleotides in length. In some instances, the nucleic acid molecule is about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 nucleotides in length. In some instances, the nucleic acid molecule is about 10 nucleotides in length. In some instances, the nucleic acid molecule is about 12 nucleotides in length. In some instances, the nucleic acid molecule is about 15 nucleotides in length. In some instances, the nucleic acid molecule is about 18 nucleotides in length. In some instances, the nucleic acid molecule is about 19 nucleotides in length. In some instances, the nucleic acid molecule is about 20 nucleotides in length. In some instances, the nucleic acid molecule is about 21 nucleotides in length. In some instances, the nucleic acid molecule is about 22 nucleotides in length. In some instances, the nucleic acid molecule is about 23 nucleotides in length. In some instances, the nucleic acid molecule is about 24 nucleotides in length. In some instances, the nucleic acid molecule is about 25 nucleotides in length. In some instances, the nucleic acid molecule is about 26 nucleotides in length. In some instances, the nucleic acid molecule is about 27 nucleotides in length. In some instances, the nucleic acid molecule is about 28 nucleotides in length. In some instances, the nucleic acid molecule is about 29 nucleotides in length. In some instances, the nucleic acid molecule is about 30 nucleotides in length. In some instances, the nucleic acid molecule is about 40 nucleotides in length. In some instances, the nucleic acid molecule is about 50 nucleotides in length. In some instances, the nucleic acid molecule is about 100 nucleotides in length. In some instances, the nucleic acid molecule is about 200 nucleotides in length. In some instances, the nucleic acid molecule is about 300 nucleotides in length. In some instances, the nucleic acid molecule is about 500 nucleotides in length. In some instances, the nucleic acid molecule is about 1000 nucleotides in length. In some instances, the nucleic acid molecule is about 2000 nucleotides in length. In some instances, the nucleic acid molecule is about 3000 nucleotides in length. In some instances, the nucleic acid molecule is about 4000 nucleotides in length. In some instances, the nucleic acid molecule is about 5000 nucleotides in length. In some instances, the nucleic acid molecule is about 8000 nucleotides in length.

In some embodiments, the nucleic acid molecule is from about 5 to about 10,000 nucleotides in length. In some instances, the nucleic acid molecule is from about 5 to about 9000 nucleotides in length, from about 5 to about 8000 nucleotides in length, from about 5 to about 7000 nucleotides in length, from about 5 to about 6000 nucleotides in length, from about 5 to about 5000 nucleotides in length, from about 5 to about 4000 nucleotides in length, from about 5 to about 3000 nucleotides in length, from about 5 to about 2000 nucleotides in length, from about 5 to about 1000 nucleotides in length, from about 5 to about 500 nucleotides in length, from about 5 to about 100 nucleotides in length, from about 5 to about 50 nucleotides in length, from about 5 to about 40 nucleotides in length, from about 5 to about 30 nucleotides in length, from about 5 to about 25 nucleotides in length, from about 5 to about 20 nucleotides in length, from about 10 to about 8000 nucleotides in length, from about 10 to about 7000 nucleotides in length, from about 10 to about 6000 nucleotides in length, from about 10 to about 5000 nucleotides in length, from about 10 to about 4000 nucleotides in length, from about 10 to about 3000 nucleotides in length, from about 10 to about 2000 nucleotides in length, from about 10 to about 1000 nucleotides in length, from about 10 to about 500 nucleotides in length, from about 10 to about 100 nucleotides in length, from about 10 to about 50 nucleotides in length, from about 10 to about 40 nucleotides in length, from about 10 to about 30 nucleotides in length, from about 10 to about 25 nucleotides in length, from about 10 to about 20 nucleotides in length, from about 18 to about 8000 nucleotides in length, from about 18 to about 7000 nucleotides in length, from about 18 to about 6000 nucleotides in length, from about 18 to about 5000 nucleotides in length, from about 18 to about 4000 nucleotides in length, from about 18 to about 3000 nucleotides in length, from about 18 to about 2000 nucleotides in length, from about 18 to about 1000 nucleotides in length, from about 18 to about 500 nucleotides in length, from about 18 to about 100 nucleotides in length, from about 18 to about 50 nucleotides in length, from about 18 to about 40 nucleotides in length, from about 18 to about 30 nucleotides in length, from about 18 to about 25 nucleotides in length, from about 12 to about 50 nucleotides in length, from about 20 to about 40 nucleotides in length, from about 20 to about 30 nucleotides in length, or from about 25 to about 30 nucleotides in length.

In some embodiments, the nucleic acid molecule comprises natural, synthetic, or artificial nucleotide analogues or bases. In some cases, the nucleic acid molecule comprises combinations of DNA, RNA and/or nucleotide analogues. In some instances, the synthetic or artificial nucleotide analogues or bases comprise modifications at one or more of ribose moiety, phosphate moiety, nucleoside moiety, or a combination thereof.

In some embodiments, a nucleotide analogue or artificial nucleotide base described above comprises a nucleic acid with a modification at a 2′ hydroxyl group of the ribose moiety. In some instances, the modification includes an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety. Exemplary alkyl moiety includes, but is not limited to, halogens, sulfurs, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols and oxygen. In some instances, the alkyl moiety further comprises a modification. In some instances, the modification comprises an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso, group, a nitrile group, a heterocycle (e.g., imidazole, hydrazino or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some instances, the alkyl moiety further comprises a hetero substitution. In some instances, the carbon of the heterocyclic group is substituted by a nitrogen, oxygen or sulfur. In some instances, the heterocyclic substitution includes but is not limited to, morpholino, imidazole, and pyrrolidino.

In some instances, the modification at the 2′ hydroxyl group is a 2′-O-methyl modification or a 2′-O-methoxyethyl (2′-O-MOE) modification. In some cases, the 2′-O-methyl modification adds a methyl group to the 2′ hydroxyl group of the ribose moiety whereas the 2′O-methoxyethyl modification adds a methoxyethyl group to the 2′ hydroxyl group of the ribose moiety.

In some instances, the modification at the 2′ hydroxyl group is a 2′-O-aminopropyl modification in which an extended amine group comprising a propyl linker binds the amine group to the 2′ oxygen. In some instances, this modification neutralizes the phosphate-derived overall negative charge of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar and thereby improves cellular uptake properties due to its zwitterionic properties.

In some instances, the modification at the 2′ hydroxyl group is a locked or bridged ribose modification (e.g., locked nucleic acid or LNA) in which the oxygen molecule bound at the 2′ carbon is linked to the 4′ carbon by a methylene group, thus forming a 2′-C,4′-C-oxy-methylene-linked bicyclic ribonucleotide monomer.

In some embodiments, additional modifications at the 2′ hydroxyl group include 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA).

In some embodiments, a nucleotide analogue comprises a modified base such as, but not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N, N-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5-(2-amino) propyl uridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2, 2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides (such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, or 6-azothymidine), 5-methyl-2-thiouridine, other thio bases (such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl groups, any O-and N-alkylated purines and pyrimidines (such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, or pyridine-2-one), phenyl and modified phenyl groups such as aminophenol or 2,4, 6-trimethoxy benzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyi nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moieties, in some cases are or are based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4′-thioribose, and other sugars, heterocycles, or carbocycles. The term nucleotide also includes universal bases. By way of example, universal bases include but are not limited to 3-nitropyrrole, 5-nitroindole, or nebularine.

In some embodiments, a nucleotide analogue further comprises a morpholino, a peptide nucleic acid (PNA), a methylphosphonate nucleotide, a thiolphosphonate nucleotide, a 2′-fluoro N3-P5′-phosphoramidite, or a 1′, 5′-anhydrohexitol nucleic acid (HNA). Morpholino or phosphorodiamidate morpholino oligo (PMO) comprises synthetic molecules whose structure mimics natural nucleic acid structure but deviates from the normal sugar and phosphate structures. In some instances, the five member ribose ring is substituted with a six member morpholino ring containing four carbons, one nitrogen, and one oxygen. In some cases, the ribose monomers are linked by a phosphordiamidate group instead of a phosphate group. In such cases, the backbone alterations remove all positive and negative charges making morpholinos neutral molecules capable of crossing cellular membranes without the aid of cellular delivery agents such as those used by charged oligonucleotides.

In some embodiments, peptide nucleic acid (PNA) does not contain sugar ring or phosphate linkage and the bases are attached and appropriately spaced by oligoglycine-like molecules, therefore, eliminating a backbone charge.

In some embodiments, one or more modifications optionally occur at the internucleotide linkage. In some instances, modified internucleotide linkage includes, but is not limited to, phosphorothioates; phosphorodithioates; methylphosphonates; 5′-alkylenephosphonates; 5′-methylphosphonate; 3′-alkylene phosphonates; borontrifluoridates; borano phosphate esters and selenophosphates of 3′-5′linkage or 2′-5′linkage; phosphotriesters; thionoalkylphosphotriesters; hydrogen phosphonate linkages; alkyl phosphonates; alkylphosphonothioates; arylphosphonothioates; phosphoroselenoates; phosphorodiselenoates; phosphinates; phosphoramidates; 3′-alkylphosphoramidates; aminoalkylphosphoramidates; thionophosphoramidates; phosphoropiperazidates; phosphoroanilothioates; phosphoroanilidates; ketones; sulfones; sulfonamides; carbonates; carbamates; methylenehydrazos; methylenedimethylhydrazos; formacetals; thioformacetals; oximes; methyleneiminos; methylenemethyliminos; thioamidates; linkages with riboacetyl groups; aminoethyl glycine; silyl or siloxane linkages; alkyl or cycloalkyl linkages with or without heteroatoms of, for example, 1 to 10 carbons that are saturated or unsaturated and/or substituted and/or contain heteroatoms; linkages with morpholino structures, amides, or polyamides wherein the bases are attached to the aza nitrogens of the backbone directly or indirectly; and combinations thereof.

In some embodiments, one or more modifications comprise a modified phosphate backbone in which the modification generates a neutral or uncharged backbone. In some instances, the phosphate backbone is modified by alkylation to generate an uncharged or neutral phosphate backbone. As used herein, alkylation includes methylation, ethylation, and propylation. In some cases, an alkyl group, as used herein in the context of alkylation, refers to a linear or branched saturated hydrocarbon group containing from 1 to 6 carbon atoms. In some instances, exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, isohexyl, 1, 1-dimethylbutyl, 2,2-dimethylbutyl, 3.3- dimethylbutyl, and 2-ethylbutyl groups. In some cases, a modified phosphate is a phosphate group as described in U.S. Pat. No. 9,481,905.

In some embodiments, additional modified phosphate backbones comprise methylphosphonate, ethylphosphonate, methylthiophosphonate, or methoxyphosphonate. In some cases, the modified phosphate is methylphosphonate. In some cases, the modified phosphate is ethylphosphonate. In some cases, the modified phosphate is methylthiophosphonate. In some cases, the modified phosphate is methoxyphosphonate.

In some embodiments, one or more modifications further optionally include modifications of the ribose moiety, phosphate backbone and the nucleoside, or modifications of the nucleotide analogues at the 3′ or the 5′ terminus. For example, the 3′ terminus optionally include a 3′ cationic group, or by inverting the nucleoside at the 3′-terminus with a 3′-3′ linkage. In another alternative, the 3′-terminus is optionally conjugated with an aminoalkyl group, e.g., a 3′ C5-aminoalkyl dT. In an additional alternative, the 3′-terminus is optionally conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site. In some instances, the 5′-terminus is conjugated with an aminoalkyl group, e.g., a 5′-O-alkylamino substituent. In some cases, the 5′-terminus is conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site.

In some embodiments, exemplary nucleic acid cargos include, but are not limited to, Fomivirsen, Mipomersen, AZD5312 (AstraZeneca), Nusinersen, and SB010 (Sterna Biologicals).

Small Molecules

In some embodiments, the cargo is a small molecule. In some instances, the small molecule is an inhibitor (e.g., a pan inhibitor or a selective inhibitor). In other instances, the small molecule is an activator. In additional cases, the small molecule is an agonist, antagonist, a partial agonist, a mixed agonist/antagonist, or a competitive antagonist.

In some embodiments, the small molecule is a drug that falls under the class of analgesics, antianxiety drugs, antiarrhythmics, antibacterials, antibiotics, anticoagulants and thrombolytics, anticonvulsants, antidepressants, antidiarrheals, antiemetics, antifungals, antihistamines, antihypertensives, anti-inflammatories, antineoplastics, antipsychotics, antipyretics, antivirals, barbiturates, beta-blockers, bronchodilators, cold cures, corticosteroids, cough suppressants, cytotoxics, decongestants, diuretics, expectorant, hormones, hypoglycemics, immunosuppressives, laxatives, muscle relaxants, sex hormones, sleeping drugs, or tranquilizers.

In some embodiments, the small molecule is an inhibitor, e.g., an inhibitor of a kinase pathway such as the Tyrosine kinase pathway or a Serine/Threonine kinase pathway. In some cases, the small molecule is a dual protein kinase inhibitor. In some cases, the small molecule is a lipid kinase inhibitor.

In some cases, the small molecule is a neuraminidase inhibitor.

In some cases, the small molecule is a carbonic anhydrase inhibitor.

In some embodiments, exemplary targets of the small molecule include, but are not limited to, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), vascular endothelial growth factor receptor 3 (VEGFR3), fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), fibroblast growth factor receptor 4 (FGFR4), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6), a receptor tyrosine kinase, a phosphoinositide 3-kinase (PI3K) isoform (e.g., PI3Kδ, also known as p110δ), Janus kinase 1 (JAK1), Janus kinase 3 (JAK3), a receptor from the family of platelet-derived growth factor receptors (PDFG-R), and carbonic anhydrase (e.g., carbonic anhydrase I).

In some embodiments, the small molecule targets a viral protein, e.g., a viral envelope protein. In some embodiments, the small molecule decreases viral adsorption to a host cell. In some embodiments, the small molecule decreases viral entry into a host cell. In some embodiments, the small molecule decreases viral replication in a host or a host cell. In some embodiments, the small molecule decreases viral assembly.

In some embodiments, exemplary small molecule cargos include, but are not limited to, lenvatinib, palbociclib, regorafenib, idelalisib, tofacitinib, nintedanib, zanamivir, ethoxzolamide, and artemisinin.

Proteins

In some embodiments, the cargo is a protein. In some instances, the protein is a full-length protein. In other instances, the protein is a fragment, e.g., a functional fragment. In some cases, the protein is a naturally occurring protein. In additional cases, the protein is a de novo engineered protein. In further cases, the protein is a fusion protein. In further cases, the protein is a recombinant protein. Exemplary proteins include, but are not limited to, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics.

In some instances, the protein is for use in an enzyme replacement therapy.

In some cases, the protein is for use in antigen production for therapeutic and/or prophylactic vaccine production. For example, the protein comprises an antigen that elicits a desirable immune response (e.g., a pro-inflammatory immune response, an anti-inflammatory immune response, an B cell response, an antibody response, a T cell response, a CD4+ T cell response, a CD8+ T cell response, a Th1 immune response, a Th2 immune response, a Th17 immune response, a Treg immune response, or a combination thereof).

In some instances, exemplary protein cargos include, but are not limited to, romiplostim, liraglutide, a human growth hormone (rHGH), human insulin (BHI), follicle-stimulating hormone (FSH), Factor VIII, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), alpha-galactosidase A, alpha-L-iduronidase, N-acetylgalactosamine-4-sulfatase, dornase alfa, tissue plasminogen activator (TPA), glucocerebrosidase, interferon-beta-1a, insulin-like growth factor 1 (IGF-1), or rasburicase.

Peptides

In some embodiments, the cargo is a peptide. In some instances, the peptide is a naturally occurring peptide. In other instances, the peptide is an artificial engineered peptide or a recombinant peptide. In some cases, the peptide targets a G-protein coupled receptor, an ion channel, a microbe, an anti-microbial target, a catalytic or other Ig-family of receptors, an intracellular target, a membrane-anchored target, or an extracellular target.

In some cases, the peptide comprises at least 2 amino acids. In some cases, the peptide comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 amino acids. In some cases, the peptide comprises at least 10 amino acids. In some cases, the peptide comprises at least 15 amino acids. In some cases, the peptide comprises at least 20 amino acids. In some cases, the peptide comprises at least 30 amino acids. In some cases, the peptide comprises at least 40 amino acids. In some cases, the peptide comprises at least 50 amino acids. In some cases, the peptide comprises at least 60 amino acids. In some cases, the peptide comprises at least 70 amino acids. In some cases, the peptide comprises at least 80 amino acids. In some cases, the peptide comprises at least 90 amino acids. In some cases, the peptide comprises at least 100 amino acids.

In some cases, the peptide comprises at most 3 amino acids. In some cases, the peptide comprises at most 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 amino acids. In some cases, the peptide comprises at most 10 amino acids. In some cases, the peptide comprises at most 15 amino acids. In some cases, the peptide comprises at most 20 amino acids. In some cases, the peptide comprises at most 30 amino acids. In some cases, the peptide comprises at most 40 amino acids. In some cases, the peptide comprises at most 50 amino acids. In some cases, the peptide comprises at most 60 amino acids. In some cases, the peptide comprises at most 70 amino acids. In some cases, the peptide comprises at most 80 amino acids. In some cases, the peptide comprises at most 90 amino acids. In some cases, the peptide comprises at most 100 amino acids.

In some cases, the peptide comprises from about 1 to about 10 kDa. In some cases, the peptide comprises from about 1 to about 9 kDa, about 1 to about 6 kDa, about 1 to about 5 kDa, about 1 to about 4 kDa, about 1 to about 3 kDa, about 2 to about 8 kDa, about 2 to about 6 kDa, about 2 to about 4 kDa, about 1.2 to about 2.8 kDa, about 1.5 to about 2.5 kDa, or about 1.5 to about 2 kDa.

In some embodiments, the peptide is a cyclic peptide. In some instances, the cyclic peptide is a macrocyclic peptide. In other instances, the cyclic peptide is a constrained peptide. The cyclic peptides are assembled with varied linkages, such as for example, head-to-tail, head-to-side-chain, side-chain-to-tail, and side-chain-to-side-chain linkages. In some instances, a cyclic peptide (e.g., a macrocyclic or a constrained peptide) has a molecular weight from about 500 Dalton to about 2000 Dalton. In other instances, a cyclic peptide (e.g., a macrocyclic or a constrained peptide) ranges from about 10 amino acids to about 100 amino acids, from about 10 amino acids to about 70 amino acids, or from about 10 amino acids to about 50 amino acids.

In some cases, the peptide is for use in antigen production for therapeutic and/or prophylactic vaccine production. For example, the peptide comprises an antigen that elicits a desirable immune response (e.g., a pro-inflammatory immune response, an anti-inflammatory immune response, an B cell response, an antibody response, a T cell response, a CD4+ T cell response, a CD8+ T cell response, a Th1 immune response, a Th2 immune response, a Th17 immune response, a Treg immune response, or a combination thereof).

In some embodiments, the peptide comprises natural amino acids, unnatural amino acids, or a combination thereof. In some instances, an amino acid residue refers to a molecule containing both an amino group and a carboxyl group. Suitable amino acids include, without limitation, both the D- and L-isomers of the naturally-occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes. The term amino acid, as used herein, includes, without limitation, α-amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.

In some instances, α-amino acid refers to a molecule containing both an amino group and a carboxyl group bound to a carbon which is designated the α-carbon.

In some instances, β-amino acid refers to a molecule containing both an amino group and a carboxyl group in a β configuration.

In some embodiments, an amino acid analog is a racemic mixture. In some instances, the D isomer of the amino acid analog is used. In some cases, the L isomer of the amino acid analog is used. In some instances, the amino acid analog comprises chiral centers that are in the R or S configuration.

In some embodiments, exemplary peptide cargos include, but are not limited to, peginesatide, insulin, adrenocorticotropic hormone (ACTH), calcitonin, oxytocin, vasopressin, octreolide, and leuprorelin.

In some embodiments, exemplary peptide cargos include, but are not limited to, Telavancin, Dalbavancin, Oritavancin, Anidulafungin, Lanreotide, Pasireotide, Romidepsin, Linaclotide, and Peginesatide.

Antibodies

In some embodiments, the cargo is an antibody or a binding fragment thereof. In some instances, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, murine antibody or binding fragment thereof, chimeric antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, bispecific antibody or biding fragment thereof, monovalent Fab′, divalent Fab₂, F(ab)′ 3 fragments, single-chain variable fragment (scFv), bis-scFv, (scFv)₂, diabody, minibody, nanobody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or binding fragment thereof, or a chemically modified derivative thereof.

In some instances, the antibody or binding fragment thereof recognizes a cell surface protein. In some instances, the cell surface protein is an antigen expressed by a cancerous cell. In some instances, the cell surface protein is a neoepitope. In some instances, the cell surface protein comprises one or more mutations compared to a wild-type protein. Exemplary cancer antigens include, but are not limited to, alpha fetoprotein, ASLG659, B7-H3, BAFF-R, Brevican, CA125 (MUC16), CA15-3, CA19-9, carcinoembryonic antigen (CEA), CA242, CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor), CTLA-4, CXCR5, E16 (LAT1, SLC7A5), FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C), epidermal growth factor, ETBR, Fc receptor-like protein 1 (FCRH1), GEDA, HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen), human chorionic gonadotropin, ICOS, IL-2 receptor, IL20Rα, Immunoglobulin superfamily receptor translocation associated 2 (IRTA2), L6, Lewis Y, Lewis X, MAGE-1, MAGE-2, MAGE-3, MAGE 4, MART1, mesothelin, MDP, MPF (SMR, MSLN), MCP1 (CCL2), macrophage inhibitory factor (MIF), MPG, MSG783, mucin, MUC1-KLH, Napi3b (SLC34A2), nectin-4, Neu oncogene product, NCA, placental alkaline phosphatase, prostate specific membrane antigen (PMSA), prostatic acid phosphatase, PSCA hlg, anti-transferrin receptor, p97, Purinergic receptor P2X ligand-gated ion channel 5 (P2X5), LY64 (Lymphocyte antigen 64 (RP105), gp100, P21, six transmembrane epithelial antigen of prostate (STEAP1), STEAP2, Sema 5b, tumor-associated glycoprotein 72 (TAG-72), TrpM4 (BR22450, F1120041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4) and the like.

In some instances, the cell surface protein comprises clusters of differentiation (CD) cell surface markers. Exemplary CD cell surface markers include, but are not limited to, CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L (L-selectin), CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD71, CD79 (e.g., CD79a, CD79b), CD90, CD95 (Fas), CD103, CD104, CD125 (IL5RA), CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), CD221, CD274, CD279 (PD-1), CD319 (SLAMF7), CD326 (EpCAM), and the like.

In some embodiments, exemplary antibodies or binding fragments thereof include, but are not limited to, zalutumumab (HuMax-EFGr, Genmab), abagovomab (Menarini), abituzumab (Merck), adecatumumab (MT201), alacizumab pegol, alemtuzumab (Campath®, MabCampath, or Campath-1H; Leukosite), AlloMune (BioTransplant), amatuximab (Morphotek, Inc.), anti-VEGF (Genetech), anatumomab mafenatox, apolizumab (hu1D10), ascrinvacumab (Pfizer Inc.), atezolizumab (MPDL3280A; Genentech/Roche), B43.13 (OvaRex, AltaRex Corporation), basiliximab (Simulect®, Novartis), belimumab (Benlysta®, GlaxoSmithKline), bevacizumab (Avastin®, Genentech), blinatumomab (Blincyto, AMG103; Amgen), BEC2 (ImGlone Systems Inc.), carlumab (Janssen Biotech), catumaxomab (Removab, Trion Pharma), CEAcide (Immunomedics), Cetuximab (Erbitux®, ImClone), citatuzumab bogatox (VB6-845), cixutumumab (IMC-A12, ImClone Systems Inc.), conatumumab (AMG 655, Amgen), dacetuzumab (SGN-40, huS2C6; Seattle Genetics, Inc.), daratumumab (Darzalex®, Janssen Biotech), detumomab, drozitumab (Genentech), durvalumab (MedImmune), dusigitumab (MedImmune), edrecolomab (MAb17-1A, Panorex, Glaxo Wellcome), elotuzumab (Empliciti™ Bristol-Myers Squibb), emibetuzumab (Eli Lilly), enavatuzumab (Facet Biotech Corp.), enfortumab vedotin (Seattle Genetics, Inc.), enoblituzumab (MGA271, MacroGenics, Inc.), ensituxumab (Neogenix Oncology, Inc.), epratuzumab (LymphoCide, Immunomedics, Inc.), ertumaxomab (Rexomun®, Trion Pharma), etaracizumab (Abegrin, MedImmune), farletuzumab (MORAb-003, Morphotek, Inc), FBTA05 (Lymphomun, Trion Pharma), ficlatuzumab (AVEO Pharmaceuticals), figitumumab (CP-751871, Pfizer), flanvotumab (ImClone Systems), fresolimumab (GC1008, Aanofi-Aventis), futuximab, glaximab, ganitumab (Amgen), girentuximab (Rencarex®, Wilex AG), IMAB362 (Claudiximab, Ganymed Pharmaceuticals AG), imalumab (Baxalta), IMC-1C11 (ImClone Systems), IMC-C225 (Imclone Systems Inc.), imgatuzumab (Genentech/Roche), intetumumab (Centocor, Inc.), ipilimumab (Yervoy®, Bristol-Myers Squibb), iratumumab (Medarex, Inc.), isatuximab (SAR650984, Sanofi-Aventis), labetuzumab (CEA-CIDE, Immunomedics), lexatumumab (ETR2-ST01, Cambridge Antibody Technology), lintuzumab (SGN-33, Seattle Genetics), lucatumumab (Novartis), lumiliximab, mapatumumab (HGS-ETR1, Human Genome Sciences), matuzumab (EMD 72000, Merck), milatuzumab (hLL1, Immunomedics, Inc.), mitumomab (BEC-2, ImClone Systems), narnatumab (ImClone Systems), necitumumab (Portrazza™, Eli Lilly), nesvacumab (Regeneron Pharmaceuticals), nimotuzumab (h-R3, BIOMAb EGFR, TheraCIM, Theraloc, or CIMAher; Biotech Pharmaceutical Co.), nivolumab (Opdivo®, Bristol-Myers Squibb), obinutuzumab (Gazyva or Gazyvaro; Hoffmann-La Roche), ocaratuzumab (AME-133v, LY2469298; Mentrik Biotech, LLC), ofatumumab (Arzerra®, Genmab), onartuzumab (Genentech), Ontuxizumab (Morphotek, Inc.), oregovomab (OvaRex®, AltaRex Corp.), otlertuzumab (Emergent BioSolutions), panitumumab (ABX-EGF, Amgen), pankomab (Glycotope GMBH), parsatuzumab (Genentech), patritumab, pembrolizumab (Keytruda®, Merck), pemtumomab (Theragyn, Antisoma), pertuzumab (Perj eta, Genentech), pidilizumab (CT-011, Medivation), polatuzumab vedotin (Genentech/Roche), pritumumab, racotumomab (Vaxira®, Recombio), ramucirumab (Cyramza®, ImClone Systems Inc.), rituximab (Rituxan®, Genentech), robatumumab (Schering-Plough), Seribantumab (Sanofi/Merrimack Pharmaceuticals, Inc.), sibrotuzumab, siltuximab (Sylvant™, Janssen Biotech), Smart MI95 (Protein Design Labs, Inc.), Smart ID10 (Protein Design Labs, Inc.), tabalumab (LY2127399, Eli Lilly), taplitumomab paptox, tenatumomab, teprotumumab (Roche), tetulomab, TGN1412 (CD28-SuperMAB or TAB08), tigatuzumab (CD-1008, Daiichi Sankyo), tositumomab, trastuzumab (Herceptin®), tremelimumab (CP-672,206; Pfizer), tucotuzumab celmoleukin (EMD Pharmaceuticals), ublituximab, urelumab (BMS-663513, Bristol-Myers Squibb), volociximab (M200, Biogen Idec), and zatuximab.

In some instances, the antibody or binding fragments thereof is an antibody-drug conjugate (ADC). In some cases, the payload of the ADC comprises, for example, but is not limited to, an auristatin derivative, maytansine, a maytansinoid, a taxane, a calicheamicin, cemadotin, a duocarmycin, a pyrrolobenzodiazepine (PDB), or a tubulysin. In some instances, the payload comprises monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). In some instances, the payload comprises DM2 (mertansine) or DM4. In some instances, the payload comprises a pyrrolobenzodiazepine dimer.

Additional Cargos

In some embodiments, the cargo is a peptidomimetic. A peptidomimetic is a small protein-like polymer designed to mimic a peptide. In some instances, the peptidomimetic comprises D-peptides. In other instances, the peptidomimetic comprises L-peptides. Exemplary peptidomimetics include peptoids and β-peptides.

In some embodiments, the cargo is a nucleotidomimetic.

Vectors and Expression Systems

In certain embodiments, the Arc polypeptides, endo-Gag polypeptides, engineered Arc and engineered endo-Gag polypeptides described supra are encoded by plasmid vectors. In some embodiments, vectors include any suitable vectors derived from either a eukaryotic or prokaryotic sources. In some cases, vectors are obtained from bacteria (e.g. E. coli), insects, yeast (e.g. Pichia pastoris), algae, or mammalian sources.

Exemplary bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pE™ vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

Exemplary insect vectors include pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT 2, or MAT vectors such as pPolh-MAT1, or pPolh-MAT2.

In some cases, yeast vectors include Gateway® pDEST™ 14 vector, Gateway® pDEST™ 15 vector, Gateway® pDEST™ 17 vector, Gateway® pDEST™ 24 vector, Gateway® pYES-DEST52 vector, pBAD-DEST49 Gateway® destination vector, pAO815 Pichia vector, pFLD1 Pichi pastoris vector, pGAPZA,B, & C Pichia pastoris vector, pPIC3.5K Pichia vector, pPIC6 A, B, & C Pichia vector, pPIC9K Pichia vector, pTEF1/Zeo, pYES2 yeast vector, pYES2/CT yeast vector, pYES2/NT A, B, & C yeast vector, or pYES3/CT yeast vector.

Exemplary algae vectors include pChlamy-4 vector or MCS vector.

Examples of mammalian vectors include transient expression vectors or stable expression vectors. Mammalian transient expression vectors include p3×FLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a,b,c, p3×FLAG-CMV 7.1, pFLAG-CMV 20, p3×FLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Mammalian stable expression vector include pFLAG-CMV 3, p3×FLAG-CMV 9, p3×FLAG-CMV 13, pFLAG-Myc-CMV 21, p3×FLAG-Myc-CMV 25, pFLAG-CMV 4, p3×FLAG-CMV 10, p3×FLAG-CMV 14, pFLAG-Myc-CMV 22, p3×FLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.

In some instances, a cell-free system is a mixture of cytoplasmic and/or nuclear components from a cell and is used for in vitro nucleic acid synthesis. In some cases, a cell-free system utilizes either prokaryotic cell components or eukaryotic cell components. Sometimes, a nucleic acid synthesis is obtained in a cell-free system based on for example Drosophila cell, Xenopus egg, or HeLa cells (ATCC® CCL-2™). Exemplary cell-free systems include, but are not limited to, E. coli S30 Extract system, E. coli T7 S30 system, or PURExpress®.

Host Cells

In some embodiments, a host cell includes any suitable cell such as a naturally derived cell or a genetically modified cell. In some instances, a host cell is a production host cell. In some instances, a host cell is a eukaryotic cell. In other instances, a host cell is a prokaryotic cell. In some cases, a eukaryotic cell includes fungi (e.g., a yeast cell), an animal cell, or a plant cell. In some cases, a prokaryotic cell is a bacterial cell. Examples of bacterial cell include gram-positive bacteria or gram-negative bacteria. In some embodiments the gram-negative bacteria is anaerobic, rod-shaped, or both.

In some instances, gram-positive bacteria include Actinobacteria, Firmicutes or Tenericutes. In some cases, gram-negative bacteria include Aquificae, Deinococcus-Thermus, Fibrobacteres-Chlorobi/Bacteroidetes (FCB group), Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes-Verrucomicrobia/Chlamydiae (PVC group), Proteobacteria, Spirochaetes or Synergistetes. In some embodiments, bacteria is Acidobacteria, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Dictyoglomi, Thermodesulfobacteria or Thermotogae. In some embodiments, a bacterial cell is Escherichia coli, Clostridium botulinum, or Coli bacilli.

Exemplary prokaryotic host cells include, but are not limited to, BL21, Mach1™ DH10B™, TOP10, DH5a, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F′, INVαF, TOP10/P3, ccdB Survival, PIR1, PIR2, Stb12™, Stb13™, or Stb14™.

In some instances, animal cells include a cell from a vertebrate or from an invertebrate. In some cases, an animal cell includes a cell from a marine invertebrate, fish, insects, amphibian, reptile, mammal, or human. In some cases, a fungus cell includes a yeast cell, such as brewer's yeast, baker's yeast, or wine yeast.

Fungi include ascomycetes such as yeast, mold, filamentous fungi, basidiomycetes, or zygomycetes. In some instances, yeast includes Ascomycota or Basidiomycota. In some cases, Ascomycota includes Saccharomycotina (true yeasts, e.g. Saccharomyces cerevisiae (baker's yeast)) or Taphrinomycotina (e.g. Schizosaccharomycetes (fission yeasts)). In some cases, Basidiomycota includes Agaricomycotina (e.g. Tremellomycetes) or Pucciniomycotina (e.g. Microbotryomycetes).

Exemplary yeast or filamentous fungi include, for example, the genus: Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kluyveromyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidi, Aspergillus, Fusarium, or Trichoderma. Exemplary yeast or filamentous fungi include, for example, the species: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Candida boidini, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Pichia metanolica, Pichia angusta, Pichia pastoris, Pichia anomala, Hansenula polymorpha, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolytica, Trichosporon pullulans, Rhodosporidium torn Aspergillus niger, Aspergillus nidulans, Aspergillus awamori, Aspergillus oryzae, Trichoderma reesei, Yarrowia lipolytica, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, Zygosaccharomyces bailii, Cryptococcus neoformans, Cryptococcus gattii, or Saccharomyces boulardii.

Exemplary yeast host cells include, but are not limited to, Pichia pastoris yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33; and Saccharomyces cerevisiae yeast strain such as INVSc1.

In some instances, additional animal cells include cells obtained from a mollusk, arthropod, annelid or sponge. In some cases, an additional animal cell is a mammalian cell, e.g., from a human, primate, ape, equine, bovine, porcine, canine, feline or rodent. In some cases, a rodent includes mouse, rat, hamster, gerbil, hamster, chinchilla, fancy rat, or guinea pig.

Exemplary mammalian host cells include, but are not limited to, 293A cell line, 293FT cell line, 293F cells, 293 H cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, Expi293F™ cells, Flp-In™ T-REx™ 293 cell line, Flp-In™-293 cell line, Flp-In™-3T3 cell line, Flp-In™-BHK cell line, Flp-In™-CHO cell line, Flp-In™-CV-1 cell line, Flp-In™-Jurkat cell line, FreeStyle™ 293-F cells, FreeStyle™ CHO-S cells, GripTite™ 293 MSR cell line, GS-CHO cell line, HepaRG™ cells, T-REx™ Jurkat cell line, Per.C6 cells, T-REx™-293 cell line, T-REx™-CHO cell line, and T-REx™-HeLa cell line.

In some instances, a mammalian host cell is a primary cell. In some instances, a mammalian host cell is a stable cell line, or a cell line that has incorporated a genetic material of interest into its own genome and has the capability to express the product of the genetic material after many generations of cell division. In some cases, a mammalian host cell is a transient cell line, or a cell line that has not incorporated a genetic material of interest into its own genome and does not have the capability to express the product of the genetic material after many generations of cell division.

Exemplary insect host cell include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expresSF+® cells.

In some instances, plant cells include a cell from algae. Exemplary insect cell lines include, but are not limited to, strains from Chlamydomonas reinhardtii 137c, or Synechococcus elongatus PPC 7942.

Methods of Use

Disclosed herein, in certain embodiments, are methods of preparing a capsid which encapsulates a cargo. In some embodiments, the method comprises incubating a plurality of Arc or endo-Gag polypeptides, engineered Arc or endo-Gag polypeptides, and/or recombinant Arc or endo-Gag polypeptides with a cargo in a solution for a time sufficient to generate a loaded Arc-based capsid or endo-Gag-based capsid.

In some instances, the method comprises mixing a solution comprising a plurality of engineered and/or recombinant Arc polypeptides with a plurality of non-Arc capsid forming subunits prior to incubating with the cargo. In some cases, the plurality of non-Arc capsid forming subunits are mixed with the plurality of engineered and/or recombinant Arc polypeptides at a ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In other cases, the plurality of non-Arc capsid forming subunits are mixed with the plurality of engineered and/or recombinant Arc polypeptides at a ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

In some cases, the time sufficient to generate a loaded Arc-based capsid or endo-Gag-based capsid is at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 10 hours, at least about 12 hours, at least about 24 hours, or more.

In some cases, the Arc-based capsid or endo-Gag-based capsid is prepared at a temperature from about 2° C. to about 37° C. In some instances, the Arc-based capsid or endo-Gag-based capsid is prepared at a temperature from about 2° C. to about 8° C., about 2° C. to about 4° C., about 20° C. to about 37° C., about 25° C. to about 37° C., about 20° C. to about 30° C., about 25° C. to about 30° C., or about 30° C. to about 37° C.

In some cases, the Arc-based capsid or endo-Gag-based capsid is prepared at room temperature.

In some instances, the Arc-based capsid or endo-Gag-based capsid is further formulated for systemic administration.

In some instances, the Arc-based capsid or endo-Gag-based capsid is further formulated for local administration.

In some instances, the Arc-based capsid or endo-Gag-based capsid is further formulated for parenteral (e.g., intra-arterial, intra-articular, intradermal, intralesional, intramuscular, intraocular, intraosseous infusion, intraperitoneal, intrathecal, intravenous, intravitreal, or subcutaneous) administration.

In some instances, the Arc-based capsid or endo-Gag-based capsid is further formulated for topical administration.

In some instances, the Arc-based capsid or endo-Gag-based capsid is further formulated for oral administration.

In some instances, the Arc-based capsid or endo-Gag-based capsid is further formulated for sublingual administration.

In some instances, the Arc-based capsid or endo-Gag-based capsid is further formulated for aerosol administration.

In certain embodiments, also described herein is a use of an Arc-based capsid or endo-Gag-based capsid for delivery of a cargo to a site of interest. In some instances, the method comprises contacting a cell at the site of interest with an Arc-based capsid or endo-Gag-based capsid for a time sufficient to facilitate cellular uptake of the capsid.

In some cases, the cell is a muscle cell, a skin cell, a blood cell, or an immune cell (e.g., a T cell or a B cell).

In some instances, the cell is a tumor cell, e.g., a solid tumor cell or a cell from a hematologic malignancy. In some cases, the solid tumor cell is a cell from a bladder cancer, breast cancer, brain cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, or thyroid cancer. In some cases, the cell from a hematologic malignancy is from a B-cell malignancy or a T-cell malignancy. In some cases, the cell is from a leukeuma, a lymphoma, a myeloma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma, mantle cell lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, peripheral T cell lymphoma, multiple myeloma, plasmacytoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), or chronic myeloid leukemia (CIVIL).

In some embodiments, the cell is a somatic cell. In some instances, the cell is a blood cell, a skin cell, a connective tissue cell, a bone cell, a muscle cell, or a cell from an organ.

In some embodiments, the cell is an epithelial cell, a connective tissue cell, a muscular cell, or a neuron.

In some instances, the cell is an endodermal cell, a mesodermal cell, or an ectodermal. In some instances, the endoderm comprises cells of the respiratory system, the intestine, the liver, the gallbladder, the pancreas, the islets of Langerhans, the thyroid, or the hindgut. In some cases, the mesoderm comprises osteochondroprogenitor cells, muscle cells, cells from the digestive system, renal stem cells, cells from the reproductive system, cells from the circulatory system (such as endothelial cells). Exemplary cells from the ectoderm comprise epithelial cells, cells of the anterior pituitary, cells of the peripheral nervous system, cells of the neuroendocrine system, cells of the eyes, cells of the central nervous system, cells of the ependymal, or cells of the pineal gland. In some cases, cells derived from the central and peripheral nervous system comprise neurons, Schwann cells, satellite glial cells, oligodendrocytes, or astrocytes. In some cases, neurons further comprise interneurons, pyramidal neurons, gabaergic neurons, dopaminergic neurons, serotoninergic neurons, glutamatergic neurons, motor neurons from the spinal cord, or inhibitory spinal neurons.

In some embodiments, the cell is a stem cell or a progenitor cell. In some cases, the cell is a mesenchymal stem or progenitor cell. In other cases, the cell is a hematopoietic stem or progenitor cell.

In some cases, a target protein is overexpressed or is depleted in the cell. In some cases, the target protein is overexpressed in the cell. In additional cases, the target protein is depleted in the cell.

In some cases, a target gene in the cell has one or more mutations.

In some cases, the cell comprises an impaired splicing mechanism.

In some instances, the Arc-based capsid is administered systemically to a subject in need thereof.

In other instances, the Arc-based capsid or endo-Gag-based capsid is administered locally to a subject in need thereof.

In some embodiments, the Arc-based capsid or endo-Gag-based capsid is administered parenterally, orally, topically, via sublingual, or by aerosol to a subject in need thereof. In some cases, the Arc-based capsid or endo-Gag-based capsid is administered parenterally to a subject in need thereof. In other cases, the Arc-based capsid or endo-Gag-based capsid is administered orally to a subject in need thereof. In additional cases, the Arc-based capsid or endo-Gag-based capsid is administered topically, via sublingual, or by aerosol to a subject in need thereof.

In some embodiments, a delivery component is combined with an Arc-based capsid or endo-Gag-based capsid for a targeted delivery to a site of interest. In some instances, the delivery component comprises a carrier, e.g., an extracellular vesicle such as a micelle, a liposome, or a microvesicle; or a viral envelope.

In some instances, the delivery component serves as a primary delivery vehicle for an Arc-based capsid or endo-Gag-based capsid which does not comprise its own delivery component (e.g., in which the second polypeptide is not present). In such cases, the delivery component directs the Arc-based capsid or endo-Gag-based capsid to a target site of interest and optionally facilitates intracellular uptake.

In other instances, the delivery component enhances target specificity and/or sensitivity of an Arc-based capsid's second polypeptide. In such cases, the delivery component enhances the specificity and/or affinity of the Arc-based capsid or endo-Gag-based capsid to the target site. In additional cases, the delivery components enhances the specificity and/or affinity by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 200-fold, 500-fold, or more. In further cases, the delivery components enhances the specificity and/or affinity by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or more. Further still, the delivery component optionally minimizes off-target effect by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 200-fold, 500-fold, or more. Further still, the delivery component optionally minimizes off-target effect by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or more.

In additional instances, the delivery component serves as a first vehicle that transports an Arc-based capsid to a general target region (e.g., a tumor microenvironment) and the Arc-based or endo-Gag-based capsid's second polypeptide serves as a second delivery molecule that drives the Arc-based capsid or endo-Gag-based capsid to the specific target site and optionally facilitates intracellular uptake. In such cases, the delivery component minimizes off-target effect by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 200-fold, 500-fold, or more. In such cases, the delivery component minimizes off-target effect by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or more.

In further instances, the delivery component serves as a first vehicle that transports an Arc-based capsid to a target site of interest and the Arc-based or endo-Gag-based capsid's second polypeptide serves as a second delivery molecule that facilitates intracellular uptake.

In some embodiments, the delivery component comprises an extracellular vesicle. In some instances, the extracellular vesicle comprises a microvesicle, a liposome, or a micelle. In some instances, the extracellular vesicle has a diameter of from about 10 nm to about 2000 nm, from about 10 nm to about 1000 nm, from about 10 nm to about 800 nm, from about 20 nm to about 600 nm, from about 30 nm to about 500 nm, from about 50 nm to about 200 nm, or from about 80 nm to about 100 nm.

In some embodiments, the delivery component comprises a microvesicle. Also known as circulating microvesicles or microparticles, microvesicles are membrane-bound vesicles that comprise phospholipids. In some instances, the microvesicle has a diameter of from about 50 nm to about 1000 nm, from about 100 nm to about 800 nm, from about 200 nm to about 500 nm, or from about 50 nm to about 400 nm.

In some instances, the microvesicle is originated from cell membrane inversion, exocytosis, shedding, blebbing, or budding. In some instances, the microvesicles are generated from differentiated cells. In other instances, the microvesicles are generated from undifferentiated cells, e.g., by blast cells, progenitor cells, or stem cells.

In some embodiments, the delivery component comprises a liposome. In some instances, the liposome comprises a plurality of lipopeptides, which are presented on the surface of the liposome, for targeted delivery to a site or region of interest. In some cases, the liposomes fuse with the target cell, whereby the contents of the liposome are then emptied into the target cell. In some cases, a liposome is endocytosed by cells that are phagocytic. Endocytosis is then followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents.

Exemplary liposomes suitable for incorporation include, and are not limited to, multilamellar vesicles (MLV), oligolamellar vesicles (OLV), unilamellar vesicles (UV), small unilamellar vesicles (SUV), medium-sized unilamellar vesicles (MUV), large unilamellar vesicles (LUV), giant unilamellar vesicles (Guy), multivesicular vesicles (MVV), single or oligolamellar vesicles made by reverse-phase evaporation method (REV), multilamellar vesicles made by the reverse-phase evaporation method (MLV-REV), stable plurilamellar vesicles (SPLV), frozen and thawed MLV (FATMLV), vesicles prepared by extrusion methods (VET), vesicles prepared by French press (FPV), vesicles prepared by fusion (FUV), dehydration-rehydration vesicles (DRV), and bubblesomes (BSV). In some instances, a liposome comprises Amphipol (A8-35). Techniques for preparing liposomes are described in, for example, COLLOIDAL DRUG DELIVERY SYSTEMS, vol. 66 (J. Kreuter ed., Marcel Dekker, Inc. (1994)).

Depending on the method of preparation, liposomes are unilamellar or multilamellar, and vary in size with diameters ranging from about 20 nm to greater than about 1000 nm.

In some instances, liposomes provided herein also comprise carrier lipids. In some embodiments the carrier lipids are phospholipids. Carrier lipids capable of forming liposomes include, but are not limited to, dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC; lecithin), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), or phosphatidylserine (PS). Other suitable phospholipids further include distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidyglycerol (DPPG), distearoylphosphatidyglycerol (DSPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidic acid (DPPA); dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dipalmitoylphosphatidylserine (DPPS), dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidyethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE) and the like, or combinations thereof. In some embodiments, the liposomes further comprise a sterol (e.g., cholesterol) which modulates liposome formation. The carrier lipids are optionally any non-phosphate polar lipids.

In some embodiments, the delivery component comprises a micelle. In some instances, the micelle has a diameter from about 2 nm to about 250 nm, from about 20 nm to about 200 nm, from about 20 nm to about 100 nm, or from about 50 to about 100 nm.

In some instances, the micelle is a polymeric micelle, characterized by a core shell structure, in which the hydrophobic core is surrounded by a hydrophilic shell. In some cases, the hydrophilic shell further comprises a hydrophilic polymer or copolymer and a pH sensitive component.

Exemplary hydrophilic polymers or copolymers include, but are not limited to, poly(N-substituted acrylamides), poly(N-acryloyl pyrrolidine), poly(N-acryloyl piperidine), poly(N-acryl-L-amino acid amides), poly(ethyl oxazoline), methylcellulose, hydroxypropyl acrylate, hydroxyalkyl cellulose derivatives and poly(vinyl alcohol), poly(N-isopropylacrylamide), poly(N-vinyl-2-pyrrolidone), polyethyleneglycol derivatives, and combinations thereof.

The pH-sensitive moiety includes, but is not limited to, an alkylacrylic acid such as methacrylic acid, ethylacrylic acid, propyl acrylic acid and butyl acrylic acid, or an amino acid such as glutamic acid.

In some instances, the hydrophobic moiety constitutes the core of the micelle and includes, for example, a single alkyl chain, such as octadecyl acrylate or a double chain alkyl compound such as phosphatidylethanolamine or dioctadecylamine. In some cases, the hydrophobic moiety is optionally a water insoluble polymer such as a poly(lactic acid) or a poly(e-caprolactone).

Polymeric micelles exhibiting pH-sensitive properties are also contemplated and are formed, e.g., by using pH-sensitive polymers including, but not limited to, copolymers from methacrylic acid, methacrylic acid esters and acrylic acid esters, polyvinyl acetate phthalate, hydroxypropyl methyl cellulose phthalate, cellulose acetate phthalate, or cellulose acetate trimellitate.

In some embodiments, the delivery component comprises a viral envelope. Viral envelopes comprise glycoproteins, phospholipids, and additional proteins obtained from a host. In some instances, the viral envelope is permissive to a wide range of target cells. In other instances, the viral envelope is non-permissive and is specific to a target cell of interest. In some cases, the viral envelope comprises a cell-specific binding protein and optionally a fusogenic molecule that aids in the fusion of the cargo into a target cell. In some cases, the viral envelope comprises an endogenous viral envelope. In other cases, the viral envelope is a modified envelop, comprising one or more foreign proteins.

In some instances, the viral envelope is derived from a DNA virus. Exemplary enveloped DNA viruses include viruses from the family of Herpesviridae, Poxviridae, and Hepadnavirdae.

In other instances, the viral envelope is derived from an RNA virus. Exemplary enveloped RNA viruses include viruses from the family of Bunyaviridae, Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, and Togaviridae.

In additional instances, the viral envelope is derived from a virus from the family of Retroviridae.

In some embodiments, the viral envelope is from an oncolytic virus, such as an oncolytic DNA virus from the family of Herpesviridae (for example, HSV1) or Poxviridae (for example, Vaccinia virus and myxoma virus); or an oncolytic RNA virus from the family of Rhabdoviridae (for example, VSV) or Paramyxoviridae (for example MV and NDV).

In some instances, the viral envelope further comprises a foreign or engineered protein that binds to an antigen or a cell surface molecule. Exemplary antigens and cell surface molecules for targeting include, but are not limited to, P-glycoprotein, Her2/Neu, erythropoietin (EPO), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGF-R), cadherin, carcinoembryonic antigen (CEA), CD4. CD8, CD19. CD20, CD33, CD34, CD45, CD117 (c-kit), CD133, HLA-A. HLA-B, HLA-C, chemokine receptor 5 (CCRS), stem cell marker ABCG2 transporter, ovarian cancer antigen CA125, immunoglobulins, integrins, prostate specific antigen (PSA), prostate stem cell antigen (PSCA), dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), thyroglobulin, granulocyte-macrophage colony stimulating factor (GM-CSF), myogenic differentiation promoting factor-1 (MyoD-1), Leu-7 (CD57), LeuM-1, cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67 (Ki-67), viral envelope proteins, HIV gp120, or transferrin receptor.

In some embodiments, the Arc-based capsid or endo-Gag-based capsid is for in vitro use.

In some instances, the Arc-based capsid or endo-Gag-based capsid is for ex vivo use.

In some cases, the Arc-based capsid or endo-Gag-based capsid is for in vivo use.

Kits/Article of Manufacture

Disclosed herein, in certain embodiments, are kits and articles of manufacture for use with one or more methods described herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic.

For example, the container(s) include a recombinant or engineered Arc or endo-Gag polypeptide described above. Such kits optionally include an identifying description or label or instructions relating to its use in the methods described herein. For example, a kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

Certain Terminologies

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood. It is to be understood that the detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

Although various features of the invention may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the invention may be described herein in the context of separate embodiments for clarity, the invention may also be implemented in a single embodiment.

Reference in the specification to “some embodiments”, “an embodiment”, “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the inventions.

As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 μL” means “about 5 μL” and also “5 μL.” Generally, the term “about” includes an amount that would be expected to be within experimental error.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

As used herein, the sequence of a CA N-lobe described herein corresponds to the human CA N-lobe. In some instances, the human CA N-lobe comprises residues 207-278 of SEQ ID NO: 1. In some instances, a CA N-lobe described herein comprises about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% sequence identity to residue 207-278 of SEQ ID NO: 1. In some cases, a CA N-lobe described herein shares a structural similarity with the human CA N-lobe. For example, a CA N-lobe described herein shares about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% structural similarity with the human CA N-lobe. In some cases, the CA N-lobe shares a high structural similarity (e.g., 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% structural similarity) but does not share a high sequence identity (e.g., the sequence identity is lower than 80%, lower than 70%, lower than 60%, lower than 50%, lower than 40%, or lower than 30%). In some cases, the CA N-lobe comprises residues 207-278 of SEQ ID NO: 1.

As used herein, the sequence of a CA C-lobe described herein corresponds to the human CA C-lobe. In some instances, the human CA C-lobe comprises residues 278-370 of SEQ ID NO: 1. In some instances, a CA C-lobe described herein comprises about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% sequence identity to residue 278-370 of SEQ ID NO: 1. In some cases, a CA C-lobe described herein shares a structural similarity with the human CA C-lobe. For example, a CA C-lobe described herein shares about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% structural similarity with the human CA C-lobe. In some cases, the CA C-lobe shares a high structural similarity (e.g., 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% structural similarity) but does not share a high sequence identity (e.g., the sequence identity is lower than 80%, lower than 70%, lower than 60%, lower than 50%, lower than 40%, or lower than 30%). In some cases, the CA C-lobe comprises residues 278-370 of SEQ ID NO: 1.

As used herein, the terms “individual(s)”, “subject(s)” and “patient(s)” mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly or a hospice worker).

EXAMPLES

These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

Example 1— Construction of DNA Vectors Encoding Recombinant Arc Proteins and Engineered Arc Proteins

To construct recombinant DNA vectors for Arc expression, full length cDNA open reading frames, excluding the initial methionine, are inserted into a cloning vector and subsequently transferred into an expression vector according to standard methods. The same approach is used to construct recombinant DNA vectors for expressing endo-Gag proteins. Human Arc cDNA includes an annotated matrix domain (MA) and a capsid domain. The capsid domain has an N-terminal lobe (NTD) and a C-terminal lobe (CTD). FIG. 1 illustrates the structure of the Human Arc protein and the predicted structure of Arc from Python, Platypus, and Orca.

cDNAs encoding engineered Arc proteins are optionally generated by recombining Arc sequences from different species (FIG. 2 ), by inserting functional domains from other proteins into an Arc protein (FIG. 3A), by modifying the sequence of an Arc protein (FIG. 3B), and/or by any combination of the approaches exemplified in FIGS. 2-3 . cDNAs encoding engineered endo-Gag proteins are likewise generated by recombining endo-Gag sequences from different species, by inserting functional domains from other proteins into an endo-Gag protein, by modifying the sequence of an endo-Gag protein, and/or by any combination of these approaches. Furthermore, an engineered endo-Gag protein optionally contains Arc sequences and an engineered Arc protein optionally contains endo-Gag sequences. Engineered Arc and endo-Gag protein monomers assemble into capsids.

cDNAs encoding the Arc and endo-Gag proteins of Table 1 were inserted into an expression vector derived from pET-41 a(+) (EMD Millipore (Novagen) Cat #70566). The entire cloning site of pET-41 a(+) was removed and replaced with the DNA having the nucleotide sequence of SEQ ID NO: 57, which encodes an alternative N-terminal tag having the amino acid sequence of SEQ ID NO: 58 and comprising a 6×His tag (SEQ ID NO: 59), a 6 amino acid spacer (SEQ ID NO: 60), and an AcTEV™ cleavage site (SEQ ID NO: 61). Arc and endo-Gag open reading frames without their starting methionine codon were inserted after the AcTEV™ cleavage site by Gibson assembly. Gibson D G, Young L, Chuang R Y, Venter J C, Hutchison C A 3rd, Smith HO (2009). “Enzymatic assembly of DNA molecules up to several hundred kilobases”. Nature Methods. 6 (5): 343-345. After expression and AcTEV™ cleavage, the N-terminus of the resulting Arc or endo-Gag protein has a single residual Glycine from the AcTEV™ cleavage site.

SEQ ID NO: 57 ATGCATCACCATCACCATCACGGCTCAGGGTCTGGTAGCGAAAATCTGT ACTTCCAGGGG SEQ ID NO: 58 MHHHHHHGSGSGSENLYFQG SEQ ID NO: 59 HHHHHH SEQ ID NO: 60 GSGSGS SEQ ID NO: 61  ENLYFQG

TABLE 1 Sequences of Arc and endo-Gag polypeptides and nucleotides. SEQ ID NO: Gene Species Amino Name Common name Proper name Sequence ID acid DNA Arc Human Homo sapiens NP_056008.1 1 29 Arc Killer Whale Orcinus orca XP_004265337.1 2 30 Arc White Tailed Deer Odocoileus XP_020755692.1 3 31 virginianus texanus Arc Platypus Ornithorhynchus XP_001512750.1 4 32 anatinus Arc Goose Anser cygnoides XP_013046406.1 5 33 domesticus Arc Dalmation Pelican Pelecanus crispus KFQ60200.1 6 34 Arc White Tailed Eagle Haliaeetus albicilla KFQ04633.1 7 35 Arc King Cobra Ophiophagus ETE60609.1 8 36 hannah Arc Ray Finned Fish Austrofundulus XP_013881732.1 9 37 limnaeus Arc Sperm Whale Physeter catodon XP_007119193.2 10 38 Arc Turkey Meleagris XP_010707654.1 11 39 gallopavo Arc Central Bearded Pogona vitticeps XP_020633722.1 12 40 Dragon Arc Chinese Alligator Alligator sinensis XP_006027442.1 13 41 Arc American Alligator Alligator XP_019337372.1 14 42 mississippiensis Arc Japanese Gekko Gekko japonicus XP_015273745.1 15 43 PNMA3 Human Homo sapiens NP_001269464.1 16 44 PNMA5 Human Homo sapiens NP_001096620.1 17 45 PNMA6A Human Homo sapiens NP_116271.3 18 46 PNMA6B Human Homo sapiens SP_P0C5W0.1 19 47 RTL3 Human Homo sapiens NP_689907.1 20 48 RTL6 Human Homo sapiens NP_115663.2 21 49 RTL8A Human Homo sapiens NP_001071640.1 22 50 RTL8B Human Homo sapiens NP_001071641.1 23 51 BOP Human Homo sapiens NP_078903.3 24 52 LDOC1 Human Homo sapiens NP_036449.1 25 53 ZNF18 Human Homo sapiens NP_001290210.1 26 54 MOAP1 Human Homo sapiens AAG31786.1 27 55 PEG10 Human Homo sapiens NP_055883.2 28 56

Example 2— Expression and Purification of Arc and Endo-Gag Proteins

Expression vectors constructs comprising Arc and endo-Gag open reading frames were transformed into the Rosetta 2 (DE3)pLysS E. coli strain (Millipore Sigma, Cat #71403). Arc or endo-Gag expression was induced with 0.1 mM IPTG followed by a 16-hour incubation at 16° C. Cell pellets were lysed by sonication in 20 mM sodium phosphate pH 7.4, 0.1M NaCl, 40 mM imidazole, 1 mM DTT, and 10% glycerol. The lysate was treated with excess TURBO DNase (Thermo Fisher Scientific, Cat # AM2238), RNase Cocktail (Thermo Fisher Scientific, Cat # AM2286), and Benzonase Nuclease (Millipore Sigma, Cat #71205) to eliminate nucleic acids. NaCl was added to lysate in order to adjust the NaCl concentration to 0.5 M followed by centrifugation and filtration to remove cellular debris. 6×His-tagged recombinant protein was loaded onto a HisTrap HP column (GE Healthcare, Cat #17-5247-01), washed with buffer A (20 mM sodium phosphate pH 7.4, 0.5M NaCl, 40 mM imidazole, and 10% glycerol), and eluted with a linear gradient of buffer B (20 mM sodium phosphate pH 7.4, 0.5M NaCl, 500 mM imidazole, and 10% glycerol). Collection tubes were supplemented in advance with 10 μl of 0.5 M EDTA pH 8.0 per 1 ml eluate. The resulting Arc or endo-Gag protein is generally more than 95% pure as revealed by SDS-PAGE analysis, with a yield of up to 50 mg per 1 L of bacterial culture. FIG. 4A.

Residual nucleic acid was removed by anion exchange chromatography on a mono Q 5/50 GL column (GE Healthcare, Cat #17516601). Before loading to the column, recombinant protein was buffer exchanged to buffer C (20 mM Tris-HCl pH 8.0, 100 mM NaCl, and 10% glycerol) using “Pierce Protein Concentrator PES, 10K MWCO, 5-20 ml” (Thermo Scientific, Cat #88528) according to the manufacturer's protocol. After loading, the mono Q resin was washed with 2 ml of buffer C. Arc and endo-Gag proteins were eluted using a linear gradient of buffer D (20 mM Tris-HCl pH 8.0, 500 mM NaCl, and 10% glycerol). RNA efficiently separated from Arc and eluted at 600 mM NaCl (FIG. 4B).

The N-terminal 6×His tag and spacer were removed from concentrating peak fractions of the mono Q purified Arc using a 10 kDa MWCO PES concentrator and then treating with 10% v/v of AcTEV™ Protease (Invitrogen™ #12575023). The cleavage efficiency is above 99% as revealed by SDS-PAGE assay. The protein is then diluted into HisTrap Buffer A and cleaned with HisTrap HP resin. The resulting purified Arc has an N-terminal Glycine residue and does not contain the initial methionine.

Example 3— Capsid Assembly

Cleaved Arc protein (1 mg/mL) was loaded into a 20 kDa MWCO dialysis cassette and dialyzed overnight in 1M sodium phosophate (pH 7.5) at room temperature. The following day, the solution was removed from the cassette, transferred to microcentrifuge tubes, and spun at max speed for 5 minutes in a tabletop centrifuge. The supernatant was transferred to a 100 kDa MWCO Regenerated Cellulose Amicon Ultrafiltration Centrifugal concentrator. The buffer was exchanged to PBS pH 7.5 and the volume was reduced 20-fold.

Capsid assembly was assayed by transmission electron microscopy. EM grids (Carbon Support Film, Square Grid, 400 mesh, 5-6 nm, Copper, CF400-Cu-UL) were prepared by glow discharge. A 5 μL sample of purified Arc was applied to the grid for 20 seconds and then wicked away using filter paper. The grid was then washed with MilliQ H₂O, stained with 54, of 1% Uranyl Acetate in H₂O for 30 seconds, and air dried for 1 minute. Images of Arc capsids were acquired using a FEI Talos L120C TEM equipped with a Gatan 4k×4k OneView camera. FIG. 5 shows concentrated human Arc capsids. FIG. 6 shows capsids formed from recombinantly expressed Arc orthologs from other vertebrate species. FIG. 7 shows capsids formed from recombinantly expressed endo-Gag genes from other vertebrate species.

Example 4— Selective Cellular Internalization of Arc Capsids

Capsids assembled from isolated recombinant human Arc protein (0.5 mg/ml) were fluorescently labeled by reacting with a 50-molar excess of NHS ester Alexa Fluor™ 594-NHS dye (Invitrogen™ #A20004) (dissolved in DMSO) in PBS (pH 8.5). Reactions were allowed to proceed for 2-hours in the dark. Alexa594-labeled capsids were then dialyzed with PBS (pH 7.5) overnight at room temperature in the dark with at least two buffer exchanges to remove any unlabeled dye.

HeLa cells (ATCC® CCL-2™) were seeded 24-hours prior to the experiment in 96-well plates at counts such that they reach ˜80% confluency for treatment. Labeled-capsids were then spiked into complete tissue culture media to a final capsid concentration of 0.05 mg/ml. Treatments proceed for 4-hours at 37° C., and then cells are washed 3-times with imaging media (DMEM, no phenol red, with 10% FBS and 20 mM HEPES) containing 10 ug/ml Hoechst nuclear stain prior to imaging. Fluorescence microscopy revealed a punctate staining pattern, suggesting that the Arc capsids were internalized by the HeLa cells (FIG. 8 ). Little or no intracellular staining was observed after administration of Alexa Fluor™ 594-labeled bovine serum albumin (BSA) (final concentration of 0.05 mg/ml) or 45.6 μM Alexa Fluor™ 594 under identical conditions.

Example 5—Heterologous RNA Delivery by Arc Capsids

Human Arc capsids were loaded with Cre RNA by spiking in excess RNA during capsid formation (by dialysis into 1M sodium phosphate). Cre RNA-loaded capsids were administered to HeLa cells in biological triplicate at a final capsid concentration of 0.05 mg/ml for 4-hours at 37° C. The cells were then washed 3-times with ice-cold 1×PBS prior to RNA extraction (Invitrogen™ TRIzol™ Reagent #15596026). Purified cell-associated RNA was quantified by qPCR in technical triplicate, normalizing values to cellular GAPDH-levels, and comparing to Escherichia coli rrsA mRNA and Arc RNA that could have carried over from protein purification. Table 2 shows primers used for the PCR reaction. The amount of cell-associated Cre RNA detected was >27-fold higher when Arc capsid were loaded with Cre RNA compared to control capsids not loaded with Cre RNA (FIG. 9 ).

TABLE 2 Primers for qPCR quantification of RNA delivered   by Arc capsids to HeLa cells SEQ ID Gene - Primer Sequence NO: GAPDH-F AAGCTCATTTCCTGGTATGACAACGA 62 GAPDH-R AGGGTCTCTCTCTTCCTCTTGTGCT 63 rrsA-F GCTCAACCTGGGAACTGCATCTGAT 64 rrsA-R TAATCCTGTTTGCTCCCCACGCTTT 65 Arc CDS-F GGCCCCTCAGCTCCAGTGATTC 66 Arc CDS-R CCTGTTGTCACTCTCCTGGCTCTGA 67 Cre CDS-F GCCAAGACATAAGAAACCTCGCCT 68 Cre CDS-R GTGAATCAACATCCTCCCTCCGTC 69

FIG. 10 illustrates an alternative method of demonstrating the delivery of a heterologous RNA by an Arc or endo-Gag capsid. 6×His-tagged Arc or endo-Gag genes are expressed in a host cell. The resulting Arc monomers are mixed with translatable Cre mRNA under capsid forming conditions to form Cre mRNA loaded capsids. Cre-loaded capsids are then administered to LoxP-luciferase reporter mice. Upon successful delivery of Cre mRNA into mouse cells and subsequent translation of Cre recombinase protein, LoxP sites of the reporter are recombined, leading to luciferase expression, which is optionally detected by bioluminescence imaging upon administration of luciferin. This method is used to test the transmission potential of candidate Arc and endo-Gag genes. A positive luciferase signal indicates that the candidate Arc or endo-Gag gene encodes an Arc or endo-Gag protein capable of assembling into capsids that incorporate a heterologous cargo and deliver that cargo to a target cell.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

TABLE 3 Arc and endo-Gag amino acid and nucleotide sequences SEQ ID NO: 1 GELDHRTSGGLHAYPGPRGGQVAKPNVILQIGKCRAEMLEHVRRTHRHLLAEVSKQVERELKGLHRSVGKLE SNLDGYVPTSDSQRWKKSIKACLCRCQETIANLERWVKREMHVWREVFYRLERWADRLESTGGKYPVGSES ARHTVSVGVGGPESYCHEADGYDYTVSPYAITPPPAAGELPGQEPAEAQQYQPWVPGEDGQPSPGVDTQI FEDPREFLSHLEEYLRQVGGSEEYWLSQIQNHMNGPAKKWWEFKQGSVKNWVEFKKEFLQYSEGTLSREAI QRELDLPQKQGEPLDQFLWRKRDLYQTLYVDADEEEIIQYVVGTLQPKLKRFLRHPLPKTLEQLIQRGMEVQ DDLEQAAEPAGPHLPVEDEAETLTPAPNSESVASDRTQPE SEQ ID NO: 2 GELDQRTTGGLHAYPAPRGGPVAKPNVILQIGKCRAEMLEHVRRTHRHLLTEVSKQVERELKGLHRSVGKLE SNLDGYVPTGDSQRWRKSIKACLCRCQETIANLERWVKREMHVWREVFYRLERWADRLESMGGKYPVGS NPSRHTTSVGVGGPESYGHEADTYDYTVSPYAITPPPAAGELPGQEAVEAQQYPPWGLGEDGQPSPGVDT QIFEDPREFLSHLEEYLRQVGGSEEYWLSQIQNHMNGPAKKWWEYKQGSVKNWVEFKKEFLQYSEGALSRE AVQRELDLPQKQGEPLDQFLWRKRDLYQTLYVDADEEEIIQYVVGTLQPKLKRFLRPPLPKTLEQLIQKGMEV EDGLEQVAEPASPHLPTEEESEALTPALTSESVASDRTQPE SEQ ID NO: 3 GELDHRTTGGLHAYPAPRGGPAAKPNVILQIGKCRAEMLEHVRRTHRHLLAEVSKQVERELKGLHRSVGKLE SNLDGYVPTGDSQRWKKSIKACLSRCQETIANLERWVKREMHVWREVFYRLERWADRLESGGGKYPVGSD PARHTVSVGVGGPESYCQDADNYDYTVSPYAITPPPAAGQLPGQEEVEAQQYPPWAPGEDGQLSPGVDTQ VFEDPREFLRHLEDYLRQVGGSEEYWLSQIQNHMNGPAKKWWEYKQGSVKNWVEFKKEFLQYSEGTLSRE AIQRELDLPQKQGEPLDQFLWRKRDLYQTLYVDAEEEEIIQYVVGTLQPKLKRFLRPPLPKTLEQLIQKGMEVQ DGLEQAAEPAAEEAEALTPALTNESVASDRTQPE SEQ ID NO: 4 GELDRLNPSSGLHPSSGLHPYPGLRGGATAKPNVILQIGKCRAEMLEHVRKTHRHLLTEVSRQVERELKGLHK SVGKLESNLDGYVPSSDSQRWKKSIKACLSRCQETIAHLERWVKREMNVWREVFYRLERWADRLEAMGGK YPAGEQARRTVSVGVGGPETCCPGDESYDCPISPYAVPPSTGESPESLDQGDQHYQQWFALPEESPVSPGV DTQIFEDPREFLRHLEKYLKQVGGTEEDWLSQIQNHMNGPAKKWWEYKQGSVKNWLEFKKEFLQYSEGTL TRDALKRELDLPQKQGEPLDQFLWRKRDLYQTLYVDADEEEIIQYVVGTLQPKLKRFLHHPLPKTLEQLIQRG QEVQNGLEPTDDPAGQRTQSEDNDESLTPAVTNESTASEGTLPE SEQ ID NO: 5 GQLDNVTNAGIHSFQGHRGVANKPNVILQIGKCRAEMLEHVRRTHRHLLSEVSKQVERELKGLQKSVGKLE NNLEDHVPTDNQRWKKSIKACLARCQETIAHLERWVKREMNVWKEVFFRLEKWADRLESMGGKYCPGEH GKQTVSVGVGGPEIRPSEGEIYDYALDMSQMYALTPPPGEMPSIPQAHDSYQWVSVSEDAPASPVETQVFE DPREFLSHLEEYLKQVGGTEEYWLSQIQNHMNGPAKKWWEYKQDSVKNWVEFKKEFLQYSEGTLTRDAIK RELDLPQKEGEPLDQFLWRKRDLYQTLYVDADEEEIIQYVVGTLQPKLKRFLSYPLPKTLEQLIQRGKEVQGN MDHSDEPSPQRTPEIQSGDSVESMPPSTTASPVPSNGTQPEPPSPPATVI SEQ ID NO: 6 GQLDNVTNAGIHSFQGHRGVANKPNVILQIGKCRAEMLEHVRRTHRHLLSEVSKQVERELKGLQKSVGKLE NNLEDHVPTDNQRWKKSIKACLARCQETIAHLERWVKREMNVWKEVFFRLEKWADRLESMGGKYCPGEH GKQTVSVGVGGPEIRPSEGEIYDYALDMSQMYALTPPPGEVPSIPQAHDSYQWVSVSEDAPASPVETQVFE DPREFLSHLEEYLKQVGGTEEYWLSQIQNHMNGPAKKWWEYKQDSVKNWVEFKKEFLQYSEGTLTRDAIK RELDLPQKEGEPLDQFLWRKRDLYQTLYVDADEEEIIQYVVGTLQPKLKRFLSYPLPKTLEQLIQRGKEVQGN MDHSEEPSPQRTPEIQSGDSVDSVPPSTTASPVPSNGTQPE SEQ ID NO: 7 GQLDNVTNAGIHSFQGHRGVANKPNVILQIGKCRAEMLEHVRRTHRHLLSEVSKQVERELKGLQKSVGKLE NNLEDHVPTDNQRWKKSIKACLARCQETIAHLERWVKREMNVWKEVFFRLEKWADRLESMGGKYCPGDH GKQTVSVGVGGPEIRPSEGEIYDYALDMSQMYALTPPPGEVPSIPQAHDSYQWVSTSEDAPASPVETQVFE DPREFLSHLEEYLKQVGGTEEYWLSQIQNHMNGPAKKWWEYKQDSVKNWVEFKKEFLQYSEGTLTRDAIK RELDLPQKEGEPLDQFLWRKRDLYQTLYVDADEEEIIQYVVGTLQPKLKRFLSYPLPKTLEQLIQRGKEVQGN MDHSEEPSPQRTPEIQSGDSVDSVPPSTTASPVPSNGTQPE SEQ ID NO: 8 GSWGLQRHVADERRGLATPTYGAVCSIREKKASQLSGQSCLEKELLGWKCTEAIVEMMQVDNFNHGNLHS CQGHRGMANHKPNVILQIGKCRAEMLDHVRRTHRHLLTEVSKQVERELKSLQKSVGKLENNLEDHVPSAAE NQRWKKSIKACLARCQETIAHLERWVKREINVWKEVFFRLEKWADRLESGGGKYGPGDQSRQTVSVGVGA PEIQPRKEEIYDYALDMSQMYALTPPPMGEDPNVPQSHDSYQWITISDDSPPSPVETQIFEDPREFLTHLEDY LKQVGGTEEYWLSQIQNHMNGPAKKWWEYKQDSVKNWLEFKKEFLQYSEGTLTRDAIKQELDLPQKDGEP LDQFLWRKRDLYQTLYIDAEEEEVIQYVVGTLQPKLKRFLSHPYPKTLEQLIQRGKEVEGNLDNSEEPSPQRSP KHQLGGSVESLPPSSTASPVASDETHPDVSAPPVTVI SEQ ID NO: 9 GDGETQAENPSTSLNNTDEDILEQLKKIVMDQQHLYQKELKASFEQLSRKMFSQMEQMNSKQTDLLLEHQ KQTVKHVDKRVEYLRAQFDASLGWRLKEQHADITTKIIPEIIQTVKEDISLCLSTLCSIAEDIQTSRATIVTGHAA VQTHPVDLLGEHHLGTTGHPRLQSTRVGKPDDVPESPVSLFMQGEARSRIVGKSPIKLQFPTFGKANDSSDP LQYLERCEDFLALNPLTDEELMATLRNVLHGTSRDWWDVARHKIQTWREFNKHFRAAFLSEDYEDELAERV RNRIQKEDESIRDFAYMYQSLCKRWNPAICEGDVVKLILKNINPQLPSQLRSRVTTVDELVRLGQQLEKDRQN QLQYELRKSSGKIIQKSSSCETSALPNTKSTPNQQNPATSNRPPQVYCWRCKGHHAPASCPQWKADKHRAQ PSRSSGPQTLTNLQAQDI SEQ ID NO: 10 GELDQRAAGGLRAYPAPRGGPVAKPSVILQIGKCRAEMLEHVRRTHRHLLTEVSKQVERELKGLHRSVGKLE GNLDGYVPTGDSQRWKKSIKACLCRCQETIANLERWVKREMHVWREVFYRLERWADRLESMGGKYPVGT NPSRHTVSVGVGGPEGYSHEADTYDYTVSPYAITPPPAAGELPGQEAVEAQQYPPWGLGEDGQPGPGVDT QIFEDPREFLSHLEEYLRQVGGSEEYWLSQIQNHMNGPAKKWWEFKQGSVKNWVEFKKEFLQYSEGTLSRE AIQRELDLPQKQGEPLDQFLWRKRDLYQTLYVDAEEEEIIQYVVGTLQPKLKRFLRPPLPKTLEQLIQKGMEVQ DGLEQAAEPASPRLPPEEESEALTPALTSESVASDRTQPE SEQ ID NO: 11 GQLDNVTNAGIHSFQGHRGVANKPNVILQIGKCRAEMLEHVRRTHRHLLSEVSKQVERELKGLQKSVGKLE NNLEDHVPTDNQRWKKSIKACLARCQETIAHLERWVKREMNVWKEVFFRLEKWADRLESMGGKYCPGEH GKQTVSVGVGGPEIRPSEGEIYDYALDMSQMYALTPGPGEVPSIPQAHDSYQWVSVSEDAPASPVETQIFED PHEFLSHLEEYLKQVGGTEEYWLSQIQNHMNGPAKKWWEYKQDSVKNWVEFKKEFLQYSEGTLTRDAIKR ELDLPQKEGEPLDQFLWRKRDLYQTLYVDADEEEIIQYVVGTLQPKLKRFLSYPLPKTLEQLIQRGKEVQGNM DHSEEPSPQRTPEIQSGDSVESMPPSTTASPVPSNGTQPEPPSPPATVI SEQ ID NO: 12 GQLENINQGSLHAFQGHRGVVHNNKPNVILQIGKCRAEMLEHVRRTHRHLLTEVSKQVERELKGLQKSVGK LENNLEDHVPSAAENQRWKKSIKACLARCQETIANLERWVKREMNVWKEVFFRLERWADRLESGGGKYCH ADQGRQTVSVGVGGPEVRPSEGEIYDYALDMSQMYALTPPPMGDVPVIPQPHDSYQWVTDPEEAPPSPV ETQIFEDPREFLTHLEDYLKQVGGTEEYWLSQIQNHMNGPAKKWWEYKQDSVKNWLEFKKEFLQYSEGTLT RDAIKQELDLPQKEGEPLDQFLWRKRDLYQTLYVEAEEEEVIQYVVGTLQPKLKRFLSHPYPKTLEQLIQRGKE VEGNLDNSEEPSPQRTPEHQLGDSVESLPPSTTASPAGSDKTQPEISLPPTTVI SEQ ID NO: 13 GQLDSVTNAGVHTYQGHRSVANKPNVILQIGKCRTEMLEHVRRTHRHLLTEVSKQVERELKGLQKSVGKLEN NLEDHVPTDNQRWKKSIKACLARCQETIAHLERWVKREMNVWKEVFFRLERWADRLESMGGKYCPTDSA RQTVSVGVGGPEIRPSEGEIYDYALDMSQMYALTPSPGELPSVPQPHDSYQWVTSPEDAPASPVETQVFED PREFLCHLEEYLKQVGGTEEYWLSQIQNHMNGPAKKWWEYKQDTVKNWVEFKKEFLQYSEGTLTRDAIKR ELDLPQKDGEPLDQFLWRKRDLYQTLYIDADEEQIIQYVVGTLQPKLKRFLSYPLPKTLEQLIQKGKEVQGSLD HSEEPSPQRASEARTGDSVETLPPSTTTSPNTSSGTQPEAPSPPATVI SEQ ID NO: 14 GQLDSVTNAGVHTYQGHRGVANKPNVILQIGKCRTEMLEHVRRTHRHLLTEVSKQVERELKGLQKSVGKLE NNLEDHVPTDNQRWKKSIKACLARCQETIAHLERWVKREMNVWKEVFFRLERWADRLESMGGKYCPTDS ARQTVSVGVGGPEIRPSEGEIYDYALDMSQMYALTPSPGELPSIPQPHDSYQWVTSPEDAPASPVETQVFED PREFLCHLEEYLKQVGGTEEYWLSQIQNHMNGPAKKWWEYKQDTVKNWVEFKKEFLQYSEGTLTRDAIKR ELDLPQKDGEPLDQFLWRKRDLYQTLYIDADEEQIIQYVVGTLQPKLKRFLSYPLPKTLEQLIQKGKEVQGSLD HSEEPSPQRASEARTGDSVESLPPSTTTSPNASSGTQPEAPSPPATVI SEQ ID NO: 15 GQLENVNHGNLHSFQGHRGGVANKPNVILQIGKCRAEMLDHVRRTHRHLLTEVSKQVERELKGLQKSVGKL ENNLEDHVPSAVENQRWKKSIKACLSRCQETIAHLERWVKREMNVWKEVFFRLERWADRLESGGGKYCHG DNHRQTVSVGVGGPEVRPSEGEIYDYALDMSQMYALTPPSPGDVPVVSQPHDSYQWVTVPEDTPPSPVET QIFEDPREFLTHLEDYLKQVGGTEEYWLSQIQNHMNGPAKKWWEYKQDSVKNWLEFKKEFLQYSEGTLTR DAIKEELDLPQKDGEPLDQFLWRKRDLYQTLYVEADEEEVIQYVVGTLQPKLKRFLSHPYPKTLEQLIQRGKEV EGNLDNSEEPTPQRTPEHQLCGSVESLPPSSTVSPVASDGTQPETSPLPATVI SEQ ID NO: 16 GPLTLLQDWCRGEHLNTRRCMLILGIPEDCGEDEFEETLQEACRHLGRYRVIGRMFRREENAQAILLELAQDI DYALLPREIPGKGGPWEVIVKPRNSDGEFLNRLNRFLEEERRTVSDMNRVLGSDTNCSAPRVTISPEFWTWA QTLGAAVQPLLEQMLYRELRVFSGNTISIPGALAFDAWLEHTTEMLQMWQVPEGEKRRRLMECLRGPALQ VVSGLRASNASITVEECLAALQQVFGPVESHKIAQVKLCKAYQEAGEKVSSFVLRLEPLLQRAVENNVVSRRN VNQTRLKRVLSGATLPDKLRDKLKLMKQRRKPPGFLALVKLLREEEEWEATLGPDRESLEGLEVAPRPPARITG VGAVPLPASGNSFDARPSQGYRRRRGRGQHRRGGVARAGSRGSRKRKRHTFCYSCGEDGHIRVQCINPSNL LLAKETKEILEGGEREAQTNSR SEQ ID NO: 17 GALTLLEDWCKGMDMDPRKALLIVGIPMECSEVEIQDTVKAGLQPLCAYRVLGRMFRREDNAKAVFIELAD TVNYTTLPSHIPGKGGSWEVVVKPRNPDDEFLSRLNYFLKDEGRSMTDVARALGCCSLPAESLDAEVMPQV RSPPLEPPKESMWYRKLKVFSGTASPSPGEETFEDWLEQVTEIMPIWQVSEVEKRRRLLESLRGPALSIMRVL QANNDSITVEQCLDALKQIFGDKEDFRASQFRFLQTSPKIGEKVSTFLLRLEPLLQKAVHKSPLSVRSTDMIRLK HLLARVAMTPALRGKLELLDQRGCPPNFLELMKLIRDEEEWENTEAVMKNKEKPSGRGRGASGRQARAEAS VSAPQATVQARSFSDSSPQTIQGGLPPLVKRRRLLGSESTRGEDHGQATYPKAENQTPGREGPQAAGEELG NEAGAGAMSHPKPWET SEQ ID NO: 18 GAVTMLQDWCRWMGVNARRGLLILGIPEDCDDAEFQESLEAALRPMGHFTVLGKAFREEDNATAALVELD REVNYALVPREIPGTGGPWNVVFVPRCSGEEFLGLGRVFHFPEQEGQMVESVAGALGVGLRRVCWLRSIG QAVQPWVEAVRCQSLGVFSGRDQPAPGEESFEVWLDHTTEMLHVWQGVSERERRRRLLEGLRGTALQLV HALLAENPARTAQDCLAALAQVFGDNESQATIRVKCLTAQQQSGERLSAFVLRLEVLLQKAMEKEALARASA DRVRLRQMLTRAHLTEPLDEALRKLRMAGRSPSFLEMLGLVRESEAWEASLARSVRAQTQEGAGARAGAQ AVARASTKVEAVPGGPGREPEGLLQAGGQEAEELLQEGLKPVLEECDN SEQ ID NO: 19 GAVTMLQDWCRWMGVNARRGLLILGIPEDCDDAEFQESLEAALRPMGHFTVLGKVFREEDNATAALVELD REVNYALVPREIPGTGGPWNVVFVPRCSGEEFLGLGRVFHFPEQEGQMVESVAGALGVGLRRVCWLRSIG QAVQPWVEAVRYQSLGVFSGRDQPAPGEESFEVWLDHTTEMLHVWQGVSERERRRRLLEGLRGTALQLV HALLAENPARTAQDCLAALAQVFGDNESQATIRVKCLTAQQQSGERLSAFVLRLEVLLQKAMEKEALARASA DRVRLRQMLTRAHLTEPLDEALRKLRMAGRSPSFLEMLGLVRESEAWEASLARSVRAQTQEGAGARAGAQ AVARASTKVEAVPGGPGREPEGLRQAGGQEAEELLQEGLKPVLEECDN SEQ ID NO: 20 GVEDLAASYIVLKLENEIRQAQVQWLMEENAALQAQIPELQKSQAAKEYDLLRKSSEAKEPQKLPEHMNPPA AWEAQKTPEFKEPQKPPEPQDLLPWEPPAAWELQEAPAAPESLAPPATRESQKPPMAHEIPTVLEGQGPA NTQDATIAQEPKNSEPQDPPNIEKPQEAPEYQETAAQLEFLELPPPQEPLEPSNAQEFLELSAAQESLEGLIVV ETSAASEFPQAPIGLEATDFPLQYTLTFSGDSQKLPEFLVQLYSYMRVRGHLYPTEAALVSFVGNCFSGRAGW WFQLLLDIQSPLLEQCESFIPVLQDTFDNPENMKDANQCIHQLCQGEGHVATHFHLIAQELNWDESTLWIQ FQEGLASSIQDELSHTSPATNLSDLITQCISLEEKPDPNPLGKSSSAEGDGPESPPAENQPMQAAINCPHISEAE WVRWHKGRLCLYCGYPGHFARDCPVKPHQALQAGNIQACQ SEQ ID NO: 21 GVQPQTSKAESPALAASPNAQMDDVIDTLTSLRLTNSALRREASTLRAEKANLTNMLESVMAELTLLRTRARI PGALQITPPISSITSNGTRPMTTPPTSLPEPFSGDPGRLAGFLMQMDRFMIFQASRFPGEAERVAFLVSRLTG EAEKWAIPHMQPDSPLRNNYQGFLAELRRTYKSPLRHARRAQIRKTSASNRAVRERQMLCRQLASAGTGPC PVHPASNGTSPAPALPARARNL SEQ ID NO: 22 GDGRVQLMKALLAGPLRPAARRWRNPIPFPETFDGDTDRLPEFIVQTSSYMFVDENTFSNDALKVTFLITRLT GPALQWVIPYIRKESPLLNDYRGFLAEMKRVFGWEEDEDF SEQ ID NO: 23 GEGRVQLMKALLARPLRPAARRWRNPIPFPETFDGDTDRLPEFIVQTSSYMFVDENTFSNDALKVTFLITRLT GPALQWVIPYIKKESPLLSDYRGFLAEMKRVFGWEEDEDF SEQ ID NO: 24 GPRGRCRQQGPRIPIWAAANYANAHPWQQMDKASPGVAYTPLVDPWIERPCCGDTVCVRTTMEQKSTA SGTCGGKPAERGPLAGHMPSSRPHRVDFCWVPGSDPGTFDGSPWLLDRFLAQLGDYMSFHFEHYQDNISR VCEILRRLTGRAQAWAAPYLDGDLPLPDDYELFCQDLKEVVQDPNSFAEYHAVVICPLPLASSQLPVAPQLP VVRQYLARFLEGLALDMGTAPRSLPAAMATPAVSGSNSVSRSALFEQQLTKESTPGPKEPPVLPSSTCSSKPG PVEPASSQPEEAAPTPVPRLSESANPPAQRPDPAHPGGPKPQKTEEEVLETEGDQEVSLGTPQEVVEAPETP GEPPLSPGF SEQ ID NO: 25 GVDELVLLLHALLMRHRALSIENSQLMEQLRLLVCERASLLRQVRPPSCPVPFPETFNGESSRLPEFIVQTASY MLVNENRFCNDAMKVAFLISLLTGEAEEWVVPYIEMDSPILGDYRAFLDEMKQCFGWDDDEDDDDEEEED DY SEQ ID NO: 26 GPVDLGQALGLLPSLAKAEDSQFSESDAALQEELSSPETARQLFRQFRYQVMSGPHETLKQLRKLCFQWLQP EVHTKEQILEILMLEQFLTILPGEIQMWVRKQCPGSGEEAVTLVESLKGDPQRLWQWISIQVLGQDILSEKME SPSCQVGEVEPHLEVVPQELGLENSSSGPGELLSHIVKEESDTEAELALAASQPARLEERLIRDQDLGASLLPAA PQEQWRQLDSTQKEQYWDLMLETYGKMVSGAGISHPKSDLTNSIEFGEELAGIYLHVNEKIPRPTCIGDRQE NDKENLNLENHRDQELLHASCQASGEVPSQASLRGFFTEDEPGCFGEGENLPEALQNIQDEGTGEQLSPQER ISEKQLGQHLPNPHSGEMSTMWLEEKRETSQKGQPRAPMAQKLPTCRECGKTFYRNSQLIFHQRTHTGETY FQCTICKKAFLRSSDFVKHQRTHTGEKPCKCDYCGKGFSDFSGLRHHEKIHTGEKPYKCPICEKSFIQRSNFNR HQRVHTGEKPYKCSHCGKSFSWSSSLDKHQRSHLGKKPFQ SEQ ID NO: 27 GTLRLLEDWCRGMDMNPRKALLIAGISQSCSVAEIEEALQAGLAPLGEYRLLGRMFRRDENRKVALVGLTAE TSHALVPKEIPGKGGIWRVIFKPPDPDNTFLSRLNEFLAGEGMTVGELSRALGHENGSLDPEQGMIPEMWA PMLAQALEALQPALQCLKYKKLRVFSGRESPEPGEEEFGRWMFHTTQMIKAWQVPDVEKRRRLLESLRGPA LDVIRVLKINNPLITVDECLQALEEVFGVTDNPRELQVKYLTTYHKDEEKLSAYVLRLEPLLQKLVQRGAIERDA VNQARLDQVIAGAVHKTIRRELNLPEDGPAPGFLQLLVLIKDYEAAEEEEALLQAILEGNFT SEQ ID NO: 28 GTERRRDELSEEINNLREKVMKQSEENNNLQSQVQKLTEENTTLREQVEPTPEDEDDDIELRGAAAAAAPPP PIEEECPEDLPEKFDGNPDMLAPFMAQCQIFMEKSTRDFSVDRVRVCFVTSMMTGRAARWASAKLERSHY LMHNYPAFMMEMKHVFEDPQRREVAKRKIRRLRQGMGSVIDYSNAFQMIAQDLDWNEPALIDQYHEGLS DHIQEELSHLEVAKSLSALIGQCIHIERRLARAAAARKPRSPPRALVLPHIASHHQVDPTEPVGGARMRLTQEE KERRRKLNLCLYCGTGGHYADNCPAKASKSSPAGKLPGPAVEGPSATGPEIIRSPQDDASSPHLQVMLQIHLP GRHTLFVRAMIDSGASGNFIDHEYVAQNGIPLRIKDWPILVEAIDGRPIASGPVVHETHDLIVDLGDHREVLSF DVTQSPFFPVVLGVRWLSTHDPNITWSTRSIVFDSEYCRYHCRMYSPIPPSLPPPAPQPPLYYPVDGYRVYQP VRYYYVQNVYTPVDEHVYPDHRLVDPHIEMIPGAHSIPSGHVYSLSEPEMAALRDFVARNVKDGLITPTIAPN GAQVLQVKRGWKLQVSYDCRAPNNFTIQNQYPRLSIPNLEDQAHLATYTEFVPQIPGYQTYPTYAAYPTYPV GFAWYPVGRDGQGRSLYVPVMITWNPHWYRQPPVPQYPPPQPPPPPPPPPPPPSYSTL SEQ ID NO: 29 GGGGAGCTGGACCACCGGACCAGCGGCGGGCTCCACGCCTACCCCGGGCCGCGGGGCGGGCAGGTGG CCAAGCCCAACGTGATCCTGCAGATCGGGAAGTGCCGGGCCGAGATGCTGGAGCACGTGCGGCGGAC GCACCGGCACCTGCTGGCCGAGGTGTCCAAGCAGGTGGAGCGCGAGCTGAAGGGGCTGCACCGGTCG GTCGGGAAGCTGGAGAGCAACCTGGACGGCTACGTGCCCACGAGCGACTCGCAGCGCTGGAAGAAGT CCATCAAGGCCTGCCTGTGCCGCTGCCAGGAGACCATCGCCAACCTGGAGCGCTGGGTCAAGCGCGAG ATGCACGTGTGGCGCGAGGTGTTCTACCGCCTGGAGCGCTGGGCCGACCGCCTGGAGTCCACGGGCGG CAAGTACCCGGTGGGCAGCGAGTCAGCCCGCCACACCGTTTCCGTGGGCGTGGGGGGTCCCGAGAGCT ACTGCCACGAGGCAGACGGCTACGACTACACCGTCAGCCCCTACGCCATCACCCCGCCCCCAGCCGCTG GCGAGCTGCCCGGGCAGGAGCCCGCCGAGGCCCAGCAGTACCAGCCGTGGGTCCCCGGCGAGGACGG GCAGCCCAGCCCCGGCGTGGACACGCAGATCTTCGAGGACCCTCGAGAGTTCCTGAGCCACCTAGAGG AGTACTTGCGGCAGGTGGGCGGCTCTGAGGAGTACTGGCTGTCCCAGATCCAGAATCACATGAACGGG CCGGCCAAGAAGTGGTGGGAGTTCAAGCAGGGCTCCGTGAAGAACTGGGTGGAGTTCAAGAAGGAGT TCCTGCAGTACAGCGAGGGCACGCTGTCCCGAGAGGCCATCCAGCGCGAGCTGGACCTGCCGCAGAAG CAGGGCGAGCCGCTGGACCAGTTCCTGTGGCGCAAGCGGGACCTGTACCAGACGCTCTACGTGGACGC GGACGAGGAGGAGATCATCCAGTACGTGGTGGGCACCCTGCAGCCCAAGCTCAAGCGTTTCCTGCGCC ACCCCCTGCCCAAGACCCTGGAGCAGCTCATCCAGAGGGGCATGGAGGTGCAGGATGACCTGGAGCAG GCGGCCGAGCCGGCCGGCCCCCACCTCCCGGTGGAGGATGAGGCGGAGACCCTCACGCCCGCCCCCAA CAGCGAGTCCGTGGCCAGTGACCGGACCCAGCCCGAG SEQ ID NO: 30 GGGGAATTGGATCAACGTACTACCGGTGGCCTTCACGCATACCCTGCACCACGCGGGGGCCCTGTCGCG AAGCCAAATGTCATCCTGCAGATTGGGAAGTGCCGGGCTGAGATGCTGGAGCACGTCCGTCGGACGCA TCGTCATCTTCTTACTGAGGTGTCAAAACAGGTGGAGCGTGAACTCAAAGGCTTGCACCGCAGCGTTGG GAAACTTGAAAGCAACTTAGATGGCTATGTGCCGACTGGCGACAGCCAGCGTTGGCGTAAGTCCATCAA AGCATGTTTGTGTCGTTGCCAGGAAACGATTGCAAACCTGGAGCGTTGGGTCAAACGGGAGATGCATG TCTGGCGTGAAGTATTTTATCGTTTAGAGCGTTGGGCCGATCGTTTAGAGAGCATGGGTGGTAAGTACC CTGTGGGGAGCAACCCTTCTCGGCATACGACGTCAGTCGGTGTTGGCGGGCCGGAGTCCTACGGTCATG AAGCGGACACCTACGACTATACCGTAAGCCCTTATGCTATTACCCCACCACCTGCGGCCGGCGAATTACC TGGCCAGGAAGCCGTTGAGGCTCAACAATACCCTCCTTGGGGGCTGGGCGAGGATGGTCAACCTAGCC CAGGGGTAGACACGCAAATCTTTGAGGACCCACGGGAGTTTCTTTCCCACCTGGAAGAATACCTGCGTC AGGTTGGTGGGAGCGAAGAATACTGGCTGTCACAAATTCAAAACCATATGAATGGTCCTGCAAAAAAAT GGTGGGAATATAAACAGGGTTCCGTGAAAAACTGGGTTGAGTTTAAAAAGGAGTTTCTTCAATATTCCG AGGGCGCCCTCAGTCGGGAGGCGGTCCAACGCGAGTTGGACTTGCCACAGAAACAGGGGGAACCACTC GATCAATTCCTTTGGCGGAAACGTGACCTTTACCAGACATTGTACGTGGATGCAGATGAGGAAGAAATT ATCCAATATGTTGTGGGGACCCTGCAGCCGAAACTGAAACGTTTCCTTCGCCCGCCGCTGCCTAAAACGT TGGAACAACTTATTCAGAAAGGTATGGAGGTCGAGGATGGCTTAGAACAAGTCGCAGAGCCGGCCTCG CCACACTTGCCTACAGAGGAGGAATCGGAGGCGCTGACCCCAGCACTTACATCAGAGTCAGTGGCATCA GACCGGACACAACCAGAG SEQ ID NO: 31 GGGGAGTTAGATCACCGTACAACGGGGGGGTTGCACGCATACCCTGCTCCACGTGGCGGGCCGGCAGC TAAGCCAAACGTAATCCTGCAGATTGGGAAGTGCCGGGCAGAGATGTTGGAGCACGTCCGGCGGACCC ACCGGCACCTCCTGGCTGAAGTGTCTAAACAAGTAGAACGGGAACTCAAAGGTCTTCATCGTAGCGTCG GGAAATTGGAATCGAATTTGGACGGGTATGTTCCTACAGGCGACTCACAGCGGTGGAAAAAGAGCATC AAGGCCTGCCTGAGTCGCTGCCAGGAGACGATTGCTAACCTCGAACGCTGGGTTAAGCGGGAGATGCA CGTTTGGCGCGAAGTCTTCTACCGGCTGGAGCGTTGGGCTGATCGGCTCGAATCTGGTGGGGGTAAGT ATCCAGTTGGGTCCGACCCTGCTCGCCACACAGTCTCAGTTGGCGTAGGTGGGCCGGAGTCGTATTGCC AAGATGCGGACAACTATGATTATACAGTTTCCCCATACGCGATCACACCACCGCCGGCAGCAGGGCAGC TGCCAGGTCAGGAAGAGGTTGAGGCCCAGCAGTATCCACCATGGGCCCCAGGGGAAGACGGCCAGCTT TCTCCTGGGGTGGACACTCAAGTTTTTGAAGATCCGCGTGAATTTCTGCGGCATTTAGAAGATTATCTCC GCCAGGTCGGGGGGTCTGAAGAGTATTGGTTAAGCCAAATTCAAAACCATATGAACGGCCCGGCCAAG AAGTGGTGGGAGTACAAGCAAGGGTCTGTGAAAAATTGGGTGGAGTTTAAGAAAGAATTCTTGCAATA TTCTGAGGGCACTCTTTCGCGTGAAGCCATCCAACGCGAACTCGACTTACCGCAGAAACAAGGGGAACC TCTCGACCAATTTCTGTGGCGCAAACGCGACCTGTACCAGACTCTTTACGTCGATGCTGAGGAGGAAGA AATTATTCAATACGTAGTTGGCACACTGCAGCCTAAGCTTAAACGGTTTTTACGTCCACCATTGCCGAAG ACGCTTGAACAACTCATCCAGAAGGGTATGGAGGTTCAAGATGGTCTGGAACAGGCAGCGGAACCAGC GGCGGAGGAGGCAGAAGCCCTGACACCTGCGTTAACTAACGAGTCTGTCGCGAGCGACCGCACCCAGC CGGAA SEQ ID NO: 32 GGGGAATTAGACCGCCTGAACCCAAGCTCAGGCCTGCATCCATCCTCTGGTTTGCATCCATACCCAGGTC TCCGGGGCGGGGCAACCGCGAAGCCTAATGTCATTTTGCAAATTGGCAAATGCCGTGCGGAAATGCTTG AACACGTCCGCAAAACTCACCGTCATCTCCTCACAGAAGTATCGCGCCAAGTAGAACGCGAGCTCAAAG GCCTTCACAAAAGTGTTGGCAAGTTGGAATCAAATCTTGATGGGTACGTACCGTCAAGCGACTCCCAAC GCTGGAAGAAAAGCATTAAGGCGTGCTTATCCCGTTGCCAAGAGACGATTGCGCATTTAGAACGCTGG GTTAAACGTGAAATGAATGTATGGCGTGAGGTGTTCTACCGTTTGGAACGTTGGGCGGACCGTCTGGA GGCTATGGGCGGTAAGTATCCTGCCGGTGAGCAGGCCCGGCGTACAGTTTCAGTGGGCGTTGGGGGCC CTGAGACATGTTGTCCAGGGGATGAAAGTTATGATTGTCCGATTTCTCCGTATGCAGTTCCACCTTCCAC CGGCGAGTCTCCGGAATCCTTAGACCAAGGGGATCAGCACTATCAGCAGTGGTTTGCCCTCCCGGAGGA GTCCCCTGTTAGCCCTGGGGTTGATACCCAGATCTTTGAAGATCCTCGCGAGTTTTTACGTCATCTGGAG AAGTACCTGAAACAAGTCGGCGGGACAGAGGAAGACTGGCTTTCTCAAATCCAGAATCACATGAATGG GCCGGCGAAGAAGTGGTGGGAGTACAAGCAAGGGAGTGTTAAGAATTGGCTTGAATTTAAGAAGGAA TTTTTACAGTATTCGGAGGGCACACTGACGCGGGACGCGTTGAAACGTGAACTGGATCTCCCACAGAAA CAAGGCGAACCACTTGATCAATTTTTATGGCGGAAGCGCGACTTATATCAGACACTCTACGTTGACGCCG ATGAAGAGGAAATCATTCAGTACGTCGTGGGCACTCTTCAGCCGAAATTAAAACGCTTTCTCCATCACCC ACTCCCTAAGACGCTTGAGCAGCTTATCCAACGGGGCCAAGAAGTTCAGAATGGTCTGGAGCCTACCGA CGATCCTGCAGGCCAACGCACTCAATCGGAGGACAACGACGAAAGCCTTACCCCTGCCGTCACCAATGA GAGTACTGCAAGCGAGGGCACCCTGCCAGAG SEQ ID NO: 33 GGGCAGCTTGATAACGTTACAAACGCGGGCATCCACTCCTTCCAGGGGCATCGTGGCGTAGCGAATAA GCCAAATGTCATTCTGCAAATTGGTAAATGTCGTGCGGAAATGCTGGAGCACGTTCGCCGCACCCACCG CCATTTATTATCTGAAGTATCTAAGCAGGTAGAACGTGAGCTGAAAGGGCTGCAAAAGTCCGTGGGCAA GCTCGAGAATAACTTGGAGGATCATGTCCCTACAGATAACCAACGCTGGAAGAAGTCCATTAAAGCGTG CTTGGCTCGTTGTCAAGAGACTATCGCGCATTTAGAGCGTTGGGTGAAACGCGAAATGAACGTCTGGAA GGAGGTGTTTTTCCGGCTGGAAAAGTGGGCAGACCGGCTGGAGTCAATGGGTGGCAAGTACTGCCCGGG CGAACACGGGAAACAAACCGTCAGTGTAGGCGTGGGGGGTCCTGAAATCCGGCCTTCGGAGGGGGA AATTTATGATTATGCTCTGGATATGAGCCAGATGTATGCACTCACCCCACCTCCAGGCGAAATGCCATCA ATCCCACAAGCCCATGACAGCTATCAGTGGGTTAGTGTCTCAGAAGATGCCCCGGCGAGCCCTGTCGAA ACCCAGGTATTTGAGGACCCTCGGGAATTCCTGTCTCACCTGGAGGAATACCTGAAGCAGGTAGGCGGC ACGGAGGAGTATTGGTTGTCCCAGATCCAGAATCACATGAATGGTCCGGCAAAAAAATGGTGGGAATA TAAACAGGACTCCGTTAAAAACTGGGTTGAGTTTAAAAAGGAATTCTTGCAATACTCTGAAGGTACTTTA ACTCGGGATGCTATTAAGCGTGAACTCGACTTGCCGCAAAAGGAAGGTGAACCTCTTGACCAATTCCTTT GGCGGAAGCGGGACCTCTATCAGACACTTTACGTGGACGCGGATGAGGAGGAGATCATTCAGTATGTG GTCGGTACCCTGCAGCCGAAGCTCAAGCGTTTCCTGAGCTATCCTCTCCCAAAGACTTTAGAACAGCTCA TCCAGCGCGGTAAAGAAGTGCAGGGTAACATGGATCACTCCGATGAGCCTTCGCCGCAGCGTACACCTG AAATTCAATCAGGTGACTCCGTAGAATCTATGCCACCTTCAACAACGGCATCTCCGGTTCCATCTAATGG TACCCAACCTGAGCCGCCGAGCCCGCCAGCCACCGTTATC SEQ ID NO: 34 GGGCAACTTGACAACGTAACAAACGCTGGGATTCACTCCTTTCAGGGCCACCGCGGTGTCGCCAACAAG CCAAACGTAATCTTGCAAATTGGCAAATGCCGTGCGGAGATGTTGGAACACGTTCGTCGTACACATCGT CACTTGCTGTCGGAAGTCTCTAAACAAGTAGAACGTGAACTTAAAGGGCTTCAAAAGTCAGTCGGCAAA TTGGAAAACAACCTTGAAGACCATGTACCAACCGACAATCAGCGTTGGAAAAAGTCTATCAAAGCTTGC CTGGCCCGTTGTCAAGAGACGATTGCTCACCTGGAGCGGTGGGTAAAGCGCGAGATGAATGTGTGGAA AGAGGTCTTCTTCCGCTTGGAAAAATGGGCCGACCGTTTGGAGTCCATGGGCGGTAAATATTGTCCGGG TGAACATGGTAAGCAAACAGTCTCTGTGGGCGTTGGTGGGCCGGAGATTCGGCCTTCTGAAGGCGAGA TTTACGATTATGCGCTCGACATGTCCCAGATGTATGCGCTTACACCACCACCGGGCGAGGTACCAAGCAT TCCTCAAGCGCATGACAGTTATCAGTGGGTTAGCGTATCCGAAGACGCTCCTGCCTCGCCGGTAGAGAC CCAGGTTTTTGAAGATCCTCGTGAATTTTTAAGCCACTTGGAGGAGTATTTGAAGCAGGTAGGGGGGAC AGAGGAATATTGGCTGTCTCAGATCCAGAACCACATGAATGGCCCGGCTAAAAAGTGGTGGGAATACA AACAAGATTCGGTAAAGAATTGGGTAGAATTTAAAAAGGAGTTTTTACAGTACTCAGAGGGGACTCTCA CGCGTGATGCGATCAAACGCGAGTTGGATCTTCCTCAAAAAGAGGGGGAGCCACTCGATCAGTTCCTCT GGCGCAAGCGGGATCTCTACCAAACACTCTACGTAGACGCAGACGAAGAAGAGATCATCCAGTACGTG GTGGGTACGCTCCAGCCGAAACTCAAACGTTTCCTCAGCTACCCACTTCCTAAGACTCTGGAACAACTGA TTCAGCGGGGCAAAGAGGTCCAGGGTAACATGGACCATTCAGAGGAACCTAGTCCGCAACGTACACCT GAGATCCAATCTGGGGATTCTGTCGATTCGGTTCCACCTTCTACAACAGCGTCTCCGGTGCCGTCAAATG GGACCCAACCAGAG SEQ ID NO: 35 GGGCAGCTTGATAATGTAACCAATGCAGGTATCCACTCTTTCCAGGGTCACCGCGGTGTGGCAAACAAG CCAAATGTTATTCTGCAAATTGGTAAGTGTCGCGCTGAGATGTTAGAACACGTCCGGCGCACGCATCGG CATCTCCTGTCAGAGGTTTCAAAGCAGGTAGAGCGTGAATTAAAGGGCCTCCAGAAGTCCGTAGGTAAA CTCGAAAATAATCTTGAAGACCACGTTCCTACCGATAATCAACGGTGGAAAAAGTCAATCAAGGCGTGC TTAGCACGGTGTCAGGAAACGATCGCGCACCTCGAACGTTGGGTGAAGCGCGAAATGAATGTCTGGAA AGAAGTGTTCTTCCGGCTTGAGAAGTGGGCTGATCGGCTCGAATCCATGGGTGGCAAATATTGTCCAGG TGATCATGGCAAGCAAACGGTCTCCGTCGGTGTTGGTGGTCCGGAAATCCGGCCGAGCGAGGGTGAAA TCTATGACTACGCTCTTGATATGTCCCAGATGTATGCACTCACTCCTCCGCCGGGTGAGGTCCCGTCGAT CCCGCAGGCGCATGACTCATACCAATGGGTGTCGACTAGCGAAGACGCACCAGCCTCCCCTGTTGAAAC TCAAGTATTCGAGGACCCGCGTGAGTTCCTGAGCCATTTAGAGGAGTACCTTAAGCAGGTTGGTGGTAC CGAGGAATACTGGTTGAGCCAGATTCAGAATCACATGAACGGGCCGGCTAAGAAATGGTGGGAATACA AGCAGGATTCAGTCAAGAATTGGGTCGAATTTAAGAAGGAGTTTTTGCAGTACAGTGAGGGGACGCTC ACACGCGACGCTATCAAACGGGAGCTGGACCTGCCACAAAAGGAGGGTGAACCGCTTGATCAGTTTCTT TGGCGCAAGCGTGATCTGTATCAAACCCTGTATGTGGACGCTGACGAAGAAGAGATCATTCAGTACGTG GTTGGGACTCTGCAACCAAAGCTGAAGCGTTTTCTTTCTTATCCTCTCCCTAAGACACTGGAACAGTTAAT CCAACGTGGCAAGGAGGTCCAGGGTAATATGGACCACTCTGAGGAACCGAGCCCGCAACGTACTCCTG AAATTCAGAGCGGGGATAGTGTCGACTCAGTTCCTCCAAGTACGACCGCATCCCCGGTCCCAAGTAACG GTACCCAACCAGAG SEQ ID NO: 36 GGGTCTTGGGGCTTGCAACGTCACGTGGCTGATGAACGTCGTGGCCTCGCTACGCCTACCTACGGCGCG GTTTGTTCCATTCGGGAGAAAAAAGCCTCCCAACTGAGCGGCCAGAGCTGTTTGGAGAAAGAGTTGCTT GGTTGGAAATGTACGGAGGCAATCGTGGAAATGATGCAAGTCGATAACTTTAACCACGGTAACTTACAT AGCTGCCAAGGCCATCGGGGGATGGCAAATCACAAACCGAACGTAATCCTTCAAATCGGGAAATGTCG CGCAGAAATGTTAGACCACGTGCGTCGCACCCACCGCCATCTCTTGACGGAGGTTTCGAAGCAGGTAGA ACGCGAATTGAAGTCTCTCCAAAAGTCGGTTGGCAAGCTCGAGAATAATCTGGAAGACCACGTGCCATC GGCAGCGGAGAACCAACGTTGGAAGAAATCAATTAAAGCCTGCCTGGCCCGGTGCCAAGAAACAATTG CTCACCTCGAACGCTGGGTTAAACGCGAAATCAACGTCTGGAAAGAAGTATTCTTTCGTCTGGAGAAGT GGGCGGACCGCCTTGAGTCGGGTGGGGGCAAGTATGGGCCTGGTGACCAAAGTCGTCAAACTGTAAGTGT CGGTGTTGGGGCCCCAGAAATCCAACCGCGGAAAGAAGAAATCTATGACTACGCTCTCGACATGTCG CAGATGTATGCCTTAACACCACCGCCGATGGGTGAAGACCCAAACGTACCTCAATCCCACGATAGCTACC AGTGGATTACCATCTCAGACGATTCACCTCCGTCGCCAGTGGAAACTCAAATTTTCGAGGATCCACGCGA ATTCCTTACCCATCTCGAGGATTATCTTAAGCAAGTGGGCGGGACTGAAGAATATTGGTTGAGTCAGATT CAAAATCATATGAACGGTCCGGCCAAGAAATGGTGGGAGTACAAACAAGATTCCGTGAAAAACTGGTT GGAATTCAAGAAGGAATTCCTTCAATACTCTGAGGGTACTTTGACACGTGACGCAATTAAACAAGAACT TGACTTACCGCAGAAGGACGGCGAGCCATTGGATCAATTTCTTTGGCGGAAGCGGGACCTGTATCAGAC GCTCTATATTGATGCAGAGGAGGAAGAAGTAATCCAATACGTTGTTGGCACACTCCAACCGAAATTAAA ACGTTTCCTTTCCCACCCGTATCCGAAAACTTTGGAACAGTTAATCCAACGTGGGAAAGAGGTGGAAGG CAACCTCGATAACTCTGAGGAGCCTAGCCCGCAACGGAGTCCAAAGCACCAATTGGGTGGTAGCGTCG AGAGCCTCCCACCTTCGTCGACCGCAAGTCCTGTTGCGTCAGACGAGACTCACCCAGACGTGAGCGCAC CTCCGGTAACGGTGATT SEQ ID NO: 37 GGGGACGGCGAGACTCAAGCTGAGAATCCATCTACCAGCTTGAACAACACTGACGAAGATATCTTGGA ACAGCTCAAGAAAATTGTCATGGATCAACAACACCTGTATCAGAAAGAATTAAAGGCATCTTTTGAACA ACTCAGTCGCAAAATGTTTTCCCAGATGGAACAAATGAATAGCAAGCAAACGGATCTGCTTTTAGAACAT CAAAAACAGACTGTCAAACATGTAGACAAGCGCGTGGAGTATTTGCGGGCGCAATTCGATGCATCGTTA GGCTGGCGGTTGAAAGAGCAACACGCGGATATTACGACCAAAATCATTCCTGAGATCATCCAAACGGTG AAGGAAGATATTAGCCTGTGTCTTTCTACGCTCTGCAGTATCGCTGAAGATATCCAGACATCACGGGCTA CCACTGTCACAGGGCATGCTGCCGTACAAACCCATCCTGTGGATCTTTTGGGTGAACACCATTTAGGGAC CACGGGGCACCCACGCTTACAGTCGACCCGTGTAGGGAAACCAGACGACGTACCTGAGTCGCCGGTAA GCCTGTTTATGCAAGGTGAGGCGCGTTCCCGGATCGTTGGCAAGAGTCCGATTAAACTGCAATTTCCGA CGTTCGGCAAAGCAAACGATTCTTCCGACCCACTCCAATATCTGGAGCGGTGTGAGGACTTTCTTGCTCT TAACCCTTTAACTGATGAGGAACTTATGGCTACTTTGCGGAATGTGTTACATGGCACCTCTCGGGATTGG TGGGATGTCGCACGTCATAAAATCCAAACTTGGCGTGAGTTTAATAAACACTTCCGGGCGGCTTTCCTCA GCGAGGATTATGAAGATGAGTTGGCTGAGCGCGTCCGTAACCGCATCCAAAAAGAAGATGAGTCTATC CGCGATTTCGCTTATATGTATCAGTCCTTGTGCAAGCGGTGGAACCCTGCTATCTGCGAAGGTGATGTAG TAAAGCTCATCCTGAAGAACATCAATCCACAACTGCCGTCTCAGTTACGCTCCCGGGTCACGACCGTGGA TGAGCTTGTTCGCTTGGGCCAGCAGCTTGAAAAAGATCGTCAGAATCAGCTCCAATATGAGCTTCGGAA GAGTTCCGGCAAAATTATCCAAAAATCTAGTTCGTGCGAAACTTCAGCGCTCCCGAACACGAAGAGTAC ACCTAATCAACAAAACCCTGCTACCAGTAACCGTCCTCCACAGGTGTATTGCTGGCGGTGTAAGGGTCAC CATGCCCCTGCCTCTTGTCCGCAATGGAAAGCTGATAAGCACCGTGCGCAACCTTCGCGGAGTTCTGGG CCACAAACTCTGACTAATCTCCAAGCTCAAGACATC SEQ ID NO: 38 GGGGAATTGGATCAACGTGCGGCAGGGGGCTTGCGCGCGTACCCGGCGCCGCGTGGTGGTCCAGTTGC CAAACCGAGCGTAATTCTTCAGATTGGTAAGTGCCGCGCTGAGATGCTGGAACACGTCCGCCGCACGCA TCGCCATCTTCTGACGGAGGTAAGTAAACAAGTGGAGCGCGAACTCAAGGGGTTACATCGGTCTGTCG GTAAGTTGGAGGGCAATTTAGACGGCTATGTGCCTACCGGTGATTCCCAACGCTGGAAAAAAAGTATCA AGGCGTGTCTCTGCCGGTGTCAGGAAACAATTGCAAATCTCGAGCGTTGGGTGAAACGTGAGATGCAT GTTTGGCGTGAGGTATTCTATCGTTTGGAACGGTGGGCAGACCGTTTGGAGTCTATGGGGGGCAAGTA TCCGGTGGGCACTAACCCGTCGCGGCACACAGTAAGTGTCGGGGTAGGGGGCCCGGAAGGCTATTCTC ATGAAGCGGATACTTATGACTACACGGTGTCTCCGTATGCTATCACGCCACCGCCTGCCGCGGGTGAGT TGCCTGGTCAAGAGGCTGTCGAGGCACAACAGTACCCTCCATGGGGTCTGGGGGAGGACGGGCAACCA GGTCCGGGCGTGGACACGCAGATTTTTGAGGACCCTCGCGAATTTTTGAGCCACTTAGAGGAGTACCTG CGGCAAGTAGGGGGGAGTGAAGAGTACTGGTTATCGCAAATTCAAAATCATATGAATGGCCCTGCGAA GAAATGGTGGGAGTTCAAACAGGGGTCAGTCAAGAATTGGGTCGAGTTTAAGAAAGAATTTTTGCAAT ACAGTGAGGGTACGTTGAGTCGCGAGGCCATCCAACGTGAACTGGACCTCCCTCAGAAGCAGGGGGAG CCGTTAGATCAATTTTTATGGCGGAAACGTGACTTATACCAAACCCTCTACGTTGACGCTGAGGAAGAA GAAATTATTCAATATGTTGTCGGTACGCTGCAGCCAAAGCTGAAGCGGTTCCTCCGTCCTCCACTCCCTA AAACCTTAGAACAATTAATCCAAAAAGGCATGGAAGTTCAGGACGGGTTAGAACAAGCGGCCGAACCG GCCTCTCCGCGTCTGCCGCCGGAAGAGGAGAGTGAGGCTCTTACGCCTGCGCTCACGAGCGAATCAGTA GCCTCCGATCGGACACAGCCAGAG SEQ ID NO: 39 GGGCAGCTTGACAATGTGACGAACGCGGGGATTCACAGCTTTCAAGGGCACCGCGGCGTCGCCAACAAA CCGAATGTCATTCTGCAAATCGGTAAATGTCGTGCTGAAATGCTTGAGCACGTTCGTCGTACCCATCGT CACTTGCTTTCTGAAGTATCAAAACAAGTGGAGCGGGAACTCAAAGGCCTGCAAAAGTCAGTGGGTAAA TTGGAGAATAACCTCGAAGACCATGTACCTACAGACAACCAGCGGTGGAAAAAATCTATCAAGGCATGC CTCGCTCGTTGCCAGGAGACTATTGCCCATCTTGAGCGGTGGGTGAAACGTGAAATGAACGTATGGAAG GAAGTATTTTTTCGCTTAGAGAAGTGGGCTGATCGTCTTGAATCGATGGGCGGCAAGTACTGTCCTGGG GAACACGGCAAACAAACTGTATCTGTCGGCGTGGGGGGCCCGGAGATCCGGCCATCGGAAGGGGAAA TTTATGATTATGCTCTCGACATGTCCCAAATGTATGCTCTCACACCAGGGCCAGGGGAAGTACCGTCAAT TCCGCAAGCACACGACAGCTACCAATGGGTATCTGTGAGCGAGGACGCGCCTGCCTCTCCGGTTGAGAC GCAAATCTTTGAGGACCCACATGAATTTTTGTCTCATCTTGAAGAATATCTCAAACAGGTTGGCGGCAC AGAAGAATACTGGTTATCTCAGATCCAGAATCACATGAACGGCCCGGCTAAAAAGTGGTGGGAGTATAA GCAAGATTCCGTAAAGAACTGGGTCGAATTCAAGAAAGAGTTTCTTCAATACTCTGAGGGTACTCTGAC GCGCGATGCAATTAAGCGGGAGTTAGACCTTCCACAAAAAGAGGGGGAGCCTCTTGACCAGTTCCTGT GGCGTAAGCGCGACCTCTATCAGACACTTTACGTCGACGCTGATGAAGAAGAGATTATTCAATATGTTG TGGGTACCCTGCAGCCAAAGCTTAAGCGTTTCCTTAGCTACCCACTTCCGAAAACTCTGGAGCAGCTCAT TCAACGCGGTAAGGAAGTGCAGGGCAACATGGACCACTCTGAAGAGCCTAGCCCGCAGCGCACTCCTG AAATCCAATCAGGTGACAGTGTGGAGTCAATGCCGCCGTCAACCACCGCTTCTCCGGTACCTAGCAACG GGACGCAACCAGAGCCTCCAAGCCCACCGGCTACAGTCATC SEQ ID NO: 40 GGGCAACTTGAGAATATTAACCAAGGTTCCCTGCACGCGTTTCAGGGTCATCGCGGCGTGGTCCATAAC AACAAGCCTAACGTTATTCTCCAGATCGGGAAGTGCCGCGCCGAAATGCTGGAGCATGTGCGGCGCACC CATCGCCATTTGCTCACTGAAGTATCAAAACAGGTGGAGCGTGAGTTGAAGGGGTTGCAGAAAAGTGT AGGCAAACTTGAAAATAATTTAGAAGACCACGTACCAAGTGCGGCTGAGAACCAACGCTGGAAGAAGT CGATTAAAGCCTGCTTAGCGCGTTGTCAGGAGACCATTGCGAACTTGGAACGCTGGGTTAAACGTGAGA TGAATGTTTGGAAGGAGGTCTTTTTCCGCTTAGAGCGCTGGGCAGATCGCCTCGAATCCGGGGGTGGCA AGTACTGCCATGCAGACCAGGGTCGCCAAACTGTCAGCGTAGGTGTTGGTGGTCCTGAAGTGCGTCCGT CTGAAGGTGAAATTTACGATTACGCGTTGGATATGAGCCAAATGTACGCCTTGACTCCGCCGCCTATGG GTGATGTTCCAGTAATTCCTCAGCCGCATGACAGTTATCAGTGGGTGACAGATCCGGAAGAAGCGCCAC CAAGTCCGGTTGAGACACAAATTTTCGAGGACCCTCGGGAGTTTCTGACCCATCTTGAGGATTATTTAAA ACAAGTCGGCGGGACAGAGGAATATTGGCTCTCACAGATCCAAAATCATATGAATGGGCCAGCGAAAA AGTGGTGGGAATATAAACAGGATAGTGTGAAGAACTGGCTTGAGTTCAAAAAAGAATTCTTGCAGTACT CAGAAGGCACGTTAACGCGGGACGCTATTAAACAGGAACTTGACCTTCCACAAAAAGAAGGGGAACCG CTGGATCAATTCCTCTGGCGCAAACGCGATTTGTACCAAACTCTCTACGTCGAGGCAGAAGAAGAGGAG GTCATCCAATATGTAGTTGGCACACTGCAACCAAAACTGAAGCGGTTTCTTTCTCATCCGTACCCTAAAA CCCTGGAGCAACTCATCCAGCGCGGGAAGGAAGTTGAGGGGAATTTGGACAATAGTGAAGAACCGTCT CCACAGCGGACCCCAGAACATCAGCTGGGGGACAGTGTGGAATCTTTGCCGCCTAGTACTACGGCTTCG CCTGCCGGTTCGGATAAAACGCAACCTGAGATTAGCTTACCTCCAACTACAGTCATT SEQ ID NO: 41 GGGCAATTAGATTCGGTAACCAATGCGGGCGTCCACACCTACCAGGGCCATCGGAGCGTCGCCAATAA ACCTAACGTCATTCTTCAAATCGGGAAATGTCGGACTGAGATGCTGGAGCATGTCCGTCGGACTCATCG CCACCTGCTCACAGAAGTGTCAAAGCAAGTGGAACGTGAACTCAAGGGCTTACAGAAGAGCGTGGGCA AACTGGAAAACAATCTTGAAGACCATGTCCCAACTGACAATCAGCGGTGGAAGAAGTCAATCAAGGCAT GTCTCGCGCGTTGCCAAGAGACCATTGCTCACCTTGAGCGGTGGGTGAAACGTGAAATGAACGTGTGG AAGGAGGTGTTCTTCCGGTTAGAACGCTGGGCCGACCGCCTTGAATCAATGGGTGGTAAATACTGCCCG ACGGACTCTGCACGTCAGACAGTTAGCGTTGGGGTGGGGGGCCCGGAAATTCGGCCTAGTGAAGGCGA AATCTATGACTACGCGCTCGATATGAGCCAAATGTACGCTCTTACGCCGTCACCGGGCGAATTGCCGTCC GTCCCTCAACCGCATGATTCATACCAGTGGGTCACTAGTCCGGAAGACGCTCCGGCGTCACCAGTTGAA ACGCAGGTATTCGAGGATCCTCGGGAGTTCTTGTGTCATTTGGAAGAGTACCTGAAGCAGGTTGGCGGTA CAGAGGAATATTGGCTGAGCCAGATTCAGAATCATATGAATGGTCCTGCAAAAAAGTGGTGGGAATA TAAACAAGACACGGTTAAGAATTGGGTGGAATTCAAGAAGGAGTTCTTACAATACAGTGAGGGTACACT TACCCGTGATGCGATTAAGCGGGAATTAGACCTCCCGCAAAAGGACGGTGAGCCTCTGGATCAATTTTT ATGGCGTAAGCGTGACCTCTATCAGACATTATACATTGATGCCGATGAAGAACAGATCATTCAGTACGTC GTGGGGACATTGCAACCTAAACTCAAGCGGTTCTTGTCCTATCCACTTCCAAAAACTCTTGAACAATTAA TCCAGAAAGGGAAGGAGGTGCAGGGTTCACTTGACCACAGCGAGGAGCCGAGTCCTCAACGTGCGAGCGAG GCTCGGACGGGCGATAGTGTGGAAACCTTGCCGCCTTCTACCACTACATCACCAAATACGTCATCT GGTACACAGCCAGAGGCACCATCGCCTCCAGCGACGGTAATC SEQ ID NO: 42 GGGCAGTTAGACAGTGTGACTAACGCCGGGGTGCATACGTACCAGGGGCACCGCGGGGTCGCCAATAA GCCAAATGTAATTCTCCAGATTGGGAAGTGTCGTACAGAGATGTTGGAACATGTCCGTCGCACTCATCG CCACTTGCTCACCGAGGTCTCCAAACAAGTAGAACGCGAACTCAAGGGGCTCCAGAAGAGTGTTGGGA AGTTGGAGAATAACCTCGAAGACCACGTTCCGACAGATAACCAACGGTGGAAAAAGTCTATTAAAGCCT GTCTCGCCCGTTGTCAAGAGACAATCGCACACTTGGAACGCTGGGTCAAACGGGAGATGAATGTGTGG AAGGAAGTCTTCTTCCGTCTCGAGCGGTGGGCGGATCGTTTAGAAAGTATGGGCGGTAAATATTGCCCA ACTGACTCGGCTCGTCAAACGGTGTCGGTTGGCGTAGGCGGCCCGGAAATTCGCCCTAGCGAGGGTGA GATCTATGACTATGCACTTGACATGAGTCAGATGTATGCGTTAACTCCGTCGCCAGGGGAGCTTCCAAGT ATTCCACAGCCTCACGATAGTTATCAATGGGTAACTTCTCCTGAAGACGCCCCAGCATCCCCAGTTGAGA CACAAGTATTCGAGGACCCTCGTGAGTTTCTCTGTCACCTCGAGGAGTACCTTAAACAGGTAGGCGGGA CCGAAGAGTACTGGTTATCGCAAATCCAAAACCATATGAATGGTCCTGCCAAAAAGTGGTGGGAGTATA AACAAGATACTGTGAAGAATTGGGTAGAGTTCAAGAAAGAGTTCTTACAGTACTCTGAGGGGACGTTA ACTCGTGATGCGATCAAGCGCGAATTGGATTTACCTCAGAAGGACGGCGAGCCACTCGACCAGTTCTTA TGGCGCAAGCGTGACTTGTATCAAACCCTTTATATCGATGCTGACGAGGAACAAATTATCCAGTACGTA GTCGGTACGTTGCAACCAAAACTTAAACGCTTTCTGAGCTACCCATTACCTAAAACGTTGGAGCAACTGA TCCAGAAAGGTAAAGAGGTGCAAGGGAGCCTGGATCATAGTGAAGAACCGAGCCCTCAGCGGGCTTCT GAAGCTCGGACCGGTGATAGCGTCGAATCTTTACCACCTAGTACCACAACCAGCCCGAATGCGTCATCT GGTACCCAACCTGAAGCGCCTTCCCCACCTGCTACAGTCATT SEQ ID NO: 43 GGGCAGCTCGAGAATGTCAACCATGGGAACCTCCATTCTTTTCAAGGTCATCGCGGCGGCGTCGCCAAC AAGCCAAACGTTATCTTGCAGATCGGTAAATGTCGTGCAGAGATGCTGGACCACGTCCGGCGGACCCAC CGGCATTTACTGACAGAGGTATCGAAACAGGTTGAACGTGAGTTGAAGGGGTTACAGAAATCAGTAGG GAAATTAGAAAATAACTTAGAAGACCATGTCCCTTCAGCCGTTGAAAACCAGCGTTGGAAAAAATCGAT CAAGGCCTGCCTTTCCCGCTGCCAAGAGACCATTGCCCACCTTGAGCGTTGGGTGAAGCGCGAGATGAA CGTATGGAAAGAGGTTTTCTTCCGCTTAGAGCGGTGGGCAGATCGGTTGGAATCTGGGGGCGGGAAAT ATTGTCACGGTGATAATCATCGTCAAACAGTATCAGTCGGTGTTGGCGGCCCTGAGGTACGTCCATCTG AAGGCGAAATTTACGATTACGCTCTCGACATGTCGCAAATGTACGCTTTAACACCGCCTAGCCCAGGGG ATGTGCCTGTAGTTAGCCAGCCGCACGACAGCTATCAGTGGGTTACGGTTCCGGAGGATACCCCTCCAT CCCCGGTGGAGACGCAAATCTTCGAGGACCCACGGGAGTTCTTGACCCACTTAGAGGATTACTTAAAGC AAGTGGGGGGTACAGAGGAATATTGGTTATCTCAGATCCAGAATCACATGAACGGGCCAGCCAAGAAG TGGTGGGAGTATAAGCAAGACTCAGTAAAAAATTGGCTCGAGTTTAAGAAGGAATTCCTTCAGTATTCC GAGGGGACACTTACGCGCGACGCTATCAAGGAAGAACTTGACCTCCCGCAAAAGGACGGGGAACCTCT TGATCAGTTCCTGTGGCGCAAGCGCGACTTGTACCAGACCCTGTACGTGGAGGCGGATGAGGAGGAGG TGATCCAGTATGTTGTGGGGACTTTACAACCTAAATTAAAGCGTTTTCTCTCACACCCTTACCCGAAAACG TTAGAGCAACTTATCCAACGGGGCAAAGAGGTGGAAGGGAACCTCGACAATTCAGAGGAACCAACACC TCAGCGTACTCCAGAACACCAACTGTGTGGTTCTGTAGAATCGCTGCCTCCTTCCTCTACCGTCAGTCCAG TGGCTAGCGATGGTACTCAACCTGAGACTTCGCCATTGCCAGCGACTGTTATT SEQ ID NO: 44 GGGCCATTGACGTTGTTACAAGACTGGTGTCGTGGTGAACATTTAAACACCCGCCGGTGCATGTTGATC CTCGGTATCCCAGAAGATTGCGGCGAGGATGAGTTCGAAGAGACACTTCAGGAGGCGTGTCGCCATTT AGGGCGGTACCGCGTGATCGGCCGCATGTTCCGTCGTGAGGAAAATGCCCAAGCGATCCTCTTGGAATT GGCGCAGGATATTGACTATGCCTTACTCCCTCGGGAAATCCCTGGGAAAGGCGGGCCTTGGGAGGTAA TTGTGAAGCCGCGTAATTCCGACGGCGAATTCTTAAATCGGCTTAATCGCTTTCTTGAAGAGGAGCGCC GTACGGTCTCCGATATGAACCGTGTTTTGGGCTCGGATACTAACTGTTCAGCTCCTCGTGTCACCATTAG TCCTGAATTCTGGACTTGGGCACAGACGCTGGGCGCAGCTGTCCAACCATTGCTCGAACAGATGCTCTA CCGGGAGTTACGGGTCTTCAGTGGCAATACGATTTCCATCCCAGGTGCTCTCGCTTTTGACGCGTGGCTG GAGCATACCACGGAAATGCTTCAAATGTGGCAGGTGCCTGAAGGGGAGAAACGGCGGCGCTTGATGG AGTGTTTGCGGGGGCCAGCCCTGCAAGTCGTTAGTGGGTTACGTGCATCGAATGCCAGTATCACTGTCG AAGAGTGTCTTGCTGCACTGCAGCAGGTATTCGGTCCAGTGGAAAGTCATAAGATTGCCCAAGTAAAGT TATGCAAAGCTTACCAGGAGGCTGGGGAAAAAGTAAGCAGCTTCGTTTTGCGTTTGGAGCCACTGCTTC AGCGTGCTGTAGAAAACAACGTGGTCAGTCGCCGCAATGTCAACCAAACACGTCTTAAGCGTGTTCTGT CGGGCGCCACCCTTCCTGACAAGCTGCGTGATAAATTGAAGTTAATGAAACAGCGCCGTAAACCGCCGG GTTTCTTGGCGTTGGTTAAACTGTTACGTGAAGAGGAGGAGTGGGAGGCCACCTTAGGGCCAGACCGC GAGTCATTGGAGGGGTTAGAAGTGGCACCGCGCCCGCCAGCACGGATTACGGGTGTTGGCGCAGTACC TCTTCCGGCATCCGGGAATTCATTTGATGCCCGTCCTTCGCAAGGGTACCGGCGCCGTCGGGGTCGTGG TCAGCACCGTCGGGGCGGCGTTGCTCGTGCAGGCTCTCGTGGCTCTCGTAAGCGGAAACGGCACACCTT CTGCTATTCCTGTGGTGAGGATGGCCATATTCGTGTCCAATGCATTAACCCTAGCAATCTCCTGTTGGCTA AGGAGACCAAAGAGATTTTGGAAGGGGGAGAACGTGAAGCGCAAACGAATTCACGT SEQ ID NO: 45 GGGGCTCTTACGCTCTTAGAAGACTGGTGTAAGGGTATGGACATGGACCCGCGGAAGGCTCTCCTGATT GTAGGTATTCCGATGGAATGCAGTGAGGTGGAAATCCAGGATACAGTTAAAGCTGGTCTTCAACCTCTG TGCGCTTATCGTGTACTCGGCCGTATGTTCCGGCGGGAGGATAATGCGAAGGCTGTTTTCATTGAGCTG GCAGACACCGTGAATTACACCACGTTACCGTCTCACATTCCGGGTAAAGGGGGTTCCTGGGAAGTCGTT GTTAAACCTCGGAACCCTGACGACGAGTTCCTTTCTCGGCTTAACTACTTCTTGAAAGATGAGGGCCGCT CGATGACGGATGTCGCCCGGGCACTGGGGTGCTGTAGCTTACCTGCGGAATCACTGGACGCGGAAGTA ATGCCACAGGTCCGCTCCCCACCATTAGAACCTCCAAAAGAGAGTATGTGGTACCGTAAGTTAAAAGTG TTTAGTGGTACCGCGTCGCCTTCGCCGGGGGAGGAGACATTTGAGGACTGGTTAGAGCAAGTCACCGA GATCATGCCTATCTGGCAAGTATCTGAAGTTGAAAAGCGCCGTCGGTTACTGGAGTCACTCCGGGGCCC GGCACTCTCAATTATGCGCGTGTTACAAGCCAATAACGATAGCATTACCGTTGAACAGTGTTTGGATGCA TTAAAGCAGATCTTTGGCGACAAGGAAGACTTCCGTGCCTCTCAATTTCGTTTTCTTCAAACGTCCCCTAA AATTGGGGAGAAGGTGAGTACGTTCCTGCTGCGTTTAGAGCCACTCTTGCAAAAGGCCGTTCACAAGAG CCCACTTTCGGTACGTAGTACTGATATGATTCGGTTAAAGCACCTGTTGGCACGCGTAGCCATGACCCCG GCACTGCGTGGTAAACTCGAATTACTCGACCAACGCGGGTGCCCACCTAATTTTCTTGAGCTGATGAAGC TGATCCGGGATGAGGAAGAGTGGGAGAATACTGAAGCTGTGATGAAAAATAAAGAGAAACCTTCAGGT CGTGGCCGCGGTGCATCAGGCCGTCAAGCTCGCGCCGAGGCCAGTGTAAGTGCTCCGCAAGCAACAGT CCAAGCACGTAGCTTCTCTGATTCTAGCCCGCAGACGATTCAGGGGGGCTTACCACCTCTTGTCAAGCGT CGGCGCCTTTTGGGTTCGGAGAGCACACGTGGGGAAGACCACGGGCAAGCTACTTATCCGAAAGCAGA GAATCAGACTCCAGGGCGTGAGGGCCCGCAGGCGGCTGGGGAGGAACTTGGTAATGAGGCCGGGGCC GGCGCGATGTCCCACCCGAAACCGTGGGAAACC SEQ ID NO: 46 GGGGCTGTGACAATGCTCCAGGACTGGTGCCGTTGGATGGGCGTGAACGCTCGGCGGGGGCTGTTAAT CTTAGGTATCCCTGAAGACTGTGACGATGCAGAGTTCCAAGAGTCGTTAGAAGCTGCACTCCGTCCTAT GGGTCACTTTACTGTACTCGGTAAGGCCTTCCGCGAGGAAGACAACGCTACCGCTGCGCTGGTGGAATT AGATCGCGAGGTTAATTACGCACTTGTTCCACGCGAAATTCCGGGCACCGGCGGGCCTTGGAACGTCGT GTTCGTTCCTCGGTGCTCCGGCGAGGAATTCCTGGGGTTAGGCCGCGTGTTCCACTTTCCTGAACAGGA GGGCCAAATGGTAGAATCGGTTGCGGGGGCACTGGGGGTAGGTCTGCGCCGCGTGTGTTGGTTACGCT CGATCGGGCAAGCTGTACAACCATGGGTAGAAGCTGTTCGCTGCCAAAGCTTAGGGGTATTTAGTGGTC GTGATCAACCTGCACCTGGTGAAGAAAGCTTCGAGGTCTGGTTGGATCATACGACCGAGATGTTGCATG TGTGGCAAGGCGTGTCGGAACGGGAACGGCGCCGTCGTCTGCTGGAAGGGCTGCGTGGCACAGCCTTA CAACTTGTACATGCCTTACTGGCAGAAAATCCGGCACGGACAGCACAAGATTGCTTGGCTGCATTAGCC CAAGTTTTTGGTGATAACGAAAGCCAGGCAACGATTCGTGTTAAATGTTTGACAGCCCAACAGCAGAGT GGCGAACGCCTCTCTGCGTTCGTTCTCCGCTTAGAAGTACTTCTGCAAAAGGCTATGGAGAAGGAAGCA TTGGCGCGCGCGTCAGCGGATCGGGTGCGTCTTCGTCAGATGCTGACACGCGCACATCTCACAGAGCCG TTGGATGAAGCCTTACGGAAATTGCGTATGGCAGGGCGTTCTCCGTCTTTTTTGGAAATGCTCGGCTTAG TACGCGAGTCAGAGGCCTGGGAGGCAAGTCTGGCTCGGTCCGTCCGGGCGCAAACCCAGGAGGGTGC AGGGGCCCGGGCGGGGGCCCAAGCAGTTGCGCGTGCCAGCACTAAGGTTGAAGCTGTACCTGGTGGC CCTGGCCGGGAGCCAGAAGGTCTCCTCCAAGCCGGGGGCCAAGAAGCGGAAGAACTTCTCCAAGAGG GCTTAAAGCCGGTTTTAGAGGAATGTGACAAT SEQ ID NO: 47 GGGGCGGTCACCATGTTGCAAGACTGGTGTCGGTGGATGGGCGTGAATGCTCGGCGGGGTTTATTGAT CTTGGGTATCCCAGAAGACTGTGACGACGCCGAGTTTCAGGAGTCGCTCGAGGCCGCCCTTCGTCCAAT GGGGCATTTTACGGTTCTGGGCAAGGTGTTCCGTGAAGAGGATAACGCTACAGCAGCTCTTGTGGAGCT TGACCGTGAGGTGAATTATGCGTTAGTACCTCGCGAGATTCCAGGTACCGGTGGGCCATGGAACGTAGT CTTCGTCCCACGTTGCTCGGGGGAGGAATTTCTGGGGCTTGGGCGCGTATTCCACTTTCCAGAACAGGA AGGGCAGATGGTCGAAAGCGTAGCAGGCGCTCTTGGCGTTGGTCTCCGGCGCGTGTGCTGGTTACGCT CCATCGGCCAAGCAGTCCAACCATGGGTTGAAGCCGTACGCTATCAATCTTTAGGTGTCTTCTCAGGCCG TGACCAGCCGGCGCCTGGTGAGGAATCCTTCGAAGTCTGGCTCGATCATACAACTGAGATGCTGCATGT ATGGCAAGGTGTCTCAGAGCGGGAACGGCGGCGGCGGTTATTAGAGGGGCTCCGTGGGACTGCGCTC CAATTAGTACATGCGCTTTTGGCCGAAAATCCAGCCCGTACTGCCCAAGATTGTCTGGCAGCACTCGCCC AAGTATTCGGCGACAACGAATCGCAGGCAACAATCCGCGTAAAGTGTCTTACAGCACAGCAGCAGTCAG GGGGAACGTCTTAGTGCGTTCTTCTGCGGCTGGAAGTGTTACTCCAGAAAGCCATGGAAAAGGAGGCA TTGGCTCGCGCGAGCGCTGACCGTGTACGTCTGCGGCAAATGCTTACTCGCGCACATCTCACCGAGCCTC TCGATGAAGCACTGCGGAAACTGCGCATGGCAGGCCGCAGCCCGTCTTTCCTGGAAATGTTAGGCTTAG TCCGGGAGTCCGAAGCCTGGGAGGCCAGTCTGGCACGGTCAGTGCGGGCACAAACGCAAGAGGGTGC AGGGGCACGGGCGGGTGCACAAGCAGTTGCACGTGCCTCCACTAAAGTTGAGGCAGTGCCGGGTGGG CCAGGCCGTGAACCGGAGGGTTTGCGCCAAGCCGGCGGGCAGGAAGCCGAAGAATTACTCCAAGAAG GTTTAAAACCGGTTTTGGAGGAATGCGATAAC SEQ ID NO: 48 GGGGTGGAAGATTTGGCGGCATCTTACATCGTATTAAAGCTTGAGAACGAAATCCGGCAGGCGCAGGT CCAATGGTTAATGGAGGAAAACGCCGCCCTGCAGGCCCAGATCCCTGAACTTCAAAAGTCGCAAGCCGC GAAGGAGTATGATCTTCTGCGTAAATCTTCGGAGGCGAAGGAGCCGCAAAAACTGCCAGAACATATGA ATCCACCGGCCGCTTGGGAAGCACAAAAGACTCCAGAGTTTAAGGAACCACAGAAACCTCCTGAACCAC AGGATTTGCTTCCTTGGGAGCCGCCTGCTGCCTGGGAGTTGCAAGAAGCACCGGCTGCCCCTGAGTCAC TGGCTCCGCCTGCAACCCGTGAGTCTCAGAAACCACCTATGGCGCATGAAATCCCTACTGTATTGGAGG GGCAAGGGCCTGCCAACACACAAGACGCTACGATTGCTCAAGAACCAAAGAATAGCGAGCCGCAAGAC CCTCCAAATATCGAGAAACCTCAGGAAGCTCCGGAATATCAAGAAACAGCGGCACAGTTGGAGTTTTTA GAACTTCCTCCACCTCAGGAGCCACTCGAACCGAGCAATGCGCAAGAATTTCTCGAGTTGTCGGCTGCCC AGGAGTCCTTAGAAGGCCTCATTGTAGTTGAAACGTCCGCGGCTTCGGAGTTCCCACAGGCTCCTATCG GGCTTGAAGCCACCGACTTTCCGCTGCAGTACACGCTTACCTTCTCTGGCGACAGCCAGAAGTTGCCAGA ATTTTTGGTCCAACTCTACAGTTATATGCGGGTACGTGGGCACTTATACCCTACCGAGGCGGCGTTAGTG TCGTTTGTAGGCAATTGTTTCTCAGGGCGCGCGGGCTGGTGGTTTCAGTTGCTTTTGGATATCCAGTCGC CTCTGTTAGAACAGTGTGAAAGTTTTATCCCGGTTCTCCAAGACACATTTGACAATCCGGAAAACATGAA GGACGCAAACCAATGCATCCACCAGCTTTGTCAGGGCGAGGGTCATGTGGCCACACACTTCCACCTCAT TGCACAAGAGCTTAATTGGGATGAAAGCACGCTGTGGATCCAGTTCCAGGAAGGCCTGGCCTCATCCAT CCAGGATGAACTTTCCCATACATCGCCTGCTACCAACCTGAGTGATCTGATTACTCAATGCATCTCATTAG AGGAAAAGCCTGACCCAAACCCGTTAGGGAAGTCCTCCTCGGCGGAGGGGGATGGCCCGGAAAGTCC GCCAGCAGAAAACCAACCTATGCAAGCTGCGATCAATTGTCCTCACATTTCCGAAGCAGAGTGGGTTCG TTGGCACAAAGGCCGGCTTTGTCTCTATTGCGGCTATCCGGGTCACTTCGCACGTGATTGCCCAGTGAAG CCACACCAGGCGTTACAGGCAGGGAACATTCAGGCTTGCCAA SEQ ID NO: 49 GGGGTGCAGCCGCAGACTAGCAAAGCTGAATCGCCGGCTCTCGCTGCCTCACCGAACGCACAAATGGA TGACGTTATTGATACATTAACCTCCCTGCGTCTGACGAATTCGGCTCTGCGGCGGGAGGCTAGCACTCTT CGGGCCGAGAAAGCAAATTTAACTAATATGCTCGAGTCAGTGATGGCCGAGTTAACGCTGTTACGGACC CGTGCGCGGATTCCGGGGGCCCTGCAGATTACGCCACCAATTTCGTCTATTACTAGCAACGGTACTCGCC CGATGACGACTCCTCCAACTAGTTTACCTGAACCGTTTTCTGGCGATCCTGGCCGGTTAGCTGGTTTCCTT ATGCAGATGGACCGTTTTATGATCTTTCAAGCTAGCCGGTTTCCAGGGGAGGCAGAGCGTGTTGCGTTC CTGGTGTCGCGCTTAACTGGCGAAGCAGAAAAATGGGCCATTCCTCACATGCAACCAGACTCTCCTTTGC GTAACAACTATCAAGGCTTCTTAGCAGAGTTACGGCGGACCTATAAGAGCCCGTTGCGTCACGCCCGGC GGGCGCAAATCCGGAAGACATCGGCCTCGAACCGGGCAGTCCGTGAACGCCAAATGCTTTGCCGGCAA CTTGCATCAGCAGGTACAGGCCCATGCCCGGTACACCCTGCTAGTAACGGGACTTCCCCGGCACCGGCA TTACCAGCACGGGCGCGTAACTTA SEQ ID NO: 50 GGGGACGGTCGGGTACAGTTGATGAAGGCTTTATTGGCTGGCCCTTTACGTCCGGCGGCACGCCGTTGG CGGAATCCTATTCCATTTCCAGAGACTTTTGATGGGGATACTGATCGCCTCCCGGAGTTTATCGTCCAA ACTTCGTCCTACATGTTCGTTGACGAAAATACTTTCTCTAACGACGCTCTGAAAGTGACATTTCTCATTAC CCGGCTGACAGGTCCAGCCTTGCAATGGGTCATTCCGTACATTCGTAAAGAAAGCCCGCTTCTTAACGAC TATCGGGGTTTCCTGGCCGAGATGAAGCGGGTTTTTGGGTGGGAAGAGGACGAGGACTTT SEQ ID NO: 51 GGGGAAGGTCGGGTGCAACTTATGAAAGCGTTGCTTGCCCGCCCGCTTCGTCCAGCAGCACGTCGCTGG CGGAATCCAATTCCTTTCCCGGAGACTTTTGACGGGGACACCGATCGGCTCCCAGAGTTCATTGTGCAGA CGTCAAGCTATATGTTCGTGGATGAGAACACGTTCTCTAACGACGCGTTGAAAGTGACTTTCTTAATTAC GCGTTTGACTGGCCCGGCTTTACAATGGGTGATTCCATACATTAAGAAAGAGTCACCGCTTCTCAGTGAT TATCGCGGTTTTTTAGCCGAGATGAAGCGGGTCTTCGGGTGGGAAGAAGACGAAGACTTT SEQ ID NO: 52 GGGCCGCGTGGGCGTTGCCGTCAACAAGGTCCTCGGATTCCGATTTGGGCAGCGGCCAACTATGCCAAC GCCCACCCGTGGCAACAAATGGATAAGGCTTCGCCAGGCGTTGCTTACACACCTTTGGTTGATCCTTGGA TTGAGCGGCCTTGTTGCGGTGACACGGTTTGTGTGCGCACCACAATGGAACAGAAGAGCACAGCGTCA GGCACTTGTGGTGGTAAGCCTGCTGAGCGTGGTCCTCTCGCGGGGCATATGCCGAGCTCACGCCCACAT CGGGTTGATTTCTGTTGGGTTCCTGGTAGCGACCCAGGCACATTCGACGGCAGTCCATGGCTCTTAGATC GCTTTTTGGCGCAACTTGGTGATTACATGAGTTTTCACTTTGAACACTACCAGGACAATATCAGCCGTGT CTGCGAGATTCTTCGTCGGTTAACGGGCCGCGCTCAGGCATGGGCTGCTCCTTACCTGGACGGGGACCT TCCACTGCCAGACGACTACGAATTGTTTTGTCAAGACCTTAAGGAGGTAGTACAGGACCCTAACAGTTTC GCCGAGTATCACGCCGTGGTGACTTGTCCACTCCCTCTTGCTTCGTCCCAACTTCCTGTAGCTCCTCAGCT TCCGGTGGTACGCCAATACCTTGCGCGCTTCTTGGAGGGCCTTGCTTTGGATATGGGTACGGCGCCTCG GTCACTCCCGGCCGCTATGGCCACACCGGCAGTCTCCGGCTCGAACTCCGTTTCTCGTTCTGCCTTATTTG AACAACAACTCACAAAGGAATCCACTCCAGGCCCGAAAGAGCCACCTGTTCTCCCTAGCTCGACTTGCTC TAGCAAACCGGGTCCTGTCGAACCAGCCAGTTCACAACCTGAAGAGGCTGCTCCTACCCCGGTGCCGCG TTTGTCAGAGTCGGCTAACCCACCGGCTCAGCGTCCAGACCCTGCTCACCCTGGTGGTCCTAAACCACAA AAAACCGAAGAGGAAGTTTTAGAAACTGAGGGGGACCAGGAAGTTAGCCTGGGGACGCCGCAGGAGG TCGTAGAAGCGCCGGAAACACCAGGTGAACCACCGCTCAGCCCTGGGTTC SEQ ID NO: 53 GGGGTTGATGAATTGGTGCTCTTGTTGCACGCGCTGTTAATGCGCCATCGGGCGCTTTCCATTGAAAATT CTCAGTTGATGGAGCAACTTCGCTTGTTGGTCTGCGAACGGGCGAGCCTTCTTCGTCAGGTACGTCCGCC GAGCTGTCCAGTGCCATTTCCTGAGACTTTTAACGGGGAGTCATCACGGTTACCTGAGTTCATCGTCCAA ACCGCAAGCTATATGTTAGTTAATGAAAATCGCTTTTGCAATGACGCAATGAAAGTCGCTTTTTTGATTA GCCTTCTTACTGGTGAAGCAGAAGAATGGGTCGTCCCATACATTGAGATGGATTCACCAATTCTTGGGG ACTACCGTGCGTTCTTGGATGAGATGAAGCAGTGTTTTGGGTGGGACGATGATGAAGATGACGACGAT GAGGAAGAGGAGGATGACTAT SEQ ID NO: 54 GGGCCTGTGGATTTAGGTCAGGCTTTGGGGTTGTTGCCATCCCTCGCTAAGGCCGAAGATTCCCAATTTAG CGAAAGCGATGCAGCTTTACAGGAGGAATTGTCTTCTCCGGAAACCGCACGGCAACTTTTTCGTCAATTTCG CTATCAAGTCATGTCGGGGCCTCATGAAACACTGAAACAGTTACGGAAGTTATGTTTTCAGTGGCTGCAAC CTGAAGTCCATACAAAGGAACAAATCCTCGAAATTCTGATGCTGGAACAGTTCTTGACCATTCTGCCTGGTG AAATTCAGATGTGGGTCCGCAAGCAGTGCCCTGGTAGTGGGGAGGAGGCGGTTACGTTAGTAGAATCCCT GAAAGGTGATCCACAACGGCTCTGGCAATGGATCTCCATCCAAGTCCTGGGTCAGGATATCCTGTCTGAGA AAATGGAGTCACCTTCTTGCCAGGTGGGCGAAGTGGAGCCACACCTGGAAGTTGTACCTCAGGAACTGGG GTTAGAGAATTCATCTTCAGGGCCGGGGGAACTTCTTTCGCACATCGTGAAAGAGGAGTCTGACACTGAAG CAGAGTTGGCGTTAGCGGCATCCCAGCCAGCTCGTTTGGAAGAACGGCTGATTCGGGATCAGGACCTTGG GGCGTCCCTCCTCCCGGCAGCACCGCAGGAGCAATGGCGTCAATTAGACAGCACTCAAAAAGAACAATATT GGGACCTGATGCTGGAGACCTACGGCAAAATGGTATCCGGCGCGGGTATCTCACACCCGAAGTCCGATTT AACGAACTCAATTGAGTTCGGTGAAGAGTTGGCAGGTATTTATTTACATGTAAACGAAAAGATTCCGCGGC CTACCTGCATTGGTGACCGCCAAGAAAACGACAAAGAAAACCTTAATTTGGAAAACCATCGTGACCAGGAA TTATTACATGCCAGCTGCCAGGCCTCGGGCGAAGTGCCATCCCAGGCATCGTTACGTGGCTTCTTTACCGAG GACGAACCTGGTTGCTTCGGCGAAGGGGAGAACCTTCCTGAGGCACTTCAGAATATCCAGGATGAGGGGA CTGGCGAACAGCTGAGCCCGCAAGAACGCATTAGTGAAAAACAGTTGGGTCAACATTTGCCAAATCCGCAC TCGGGGGAGATGTCGACGATGTGGCTTGAAGAAAAACGGGAGACCAGCCAGAAAGGCCAACCACGTGCA CCAATGGCGCAGAAATTGCCAACGTGCCGCGAATGTGGCAAAACGTTTTATCGCAATAGTCAACTTATCTTT CACCAACGCACACACACCGGTGAGACATATTTTCAATGCACCATCTGCAAAAAGGCGTTTCTCCGGTCATCT GATTTCGTGAAACATCAGCGGACTCATACTGGCGAAAAACCTTGTAAATGTGACTATTGTGGCAAGGGCTT TAGTGATTTTAGCGGGCTTCGGCATCACGAGAAGATCCATACCGGCGAGAAGCCATACAAGTGTCCAATCT GTGAGAAATCTTTCATCCAGCGCAGTAATTTTAACCGCCACCAACGGGTTCACACCGGTGAAAAGCCTTATA AATGCTCGCATTGTGGCAAGAGCTTCAGCTGGAGCTCCTCGCTCGATAAGCATCAACGTTCACATCTGGGG AAGAAGCCGTTCCAA SEQ ID NO: 55 GGGACTCTCCGCTTACTTGAGGATTGGTGTCGGGGGATGGACATGAACCCACGTAAGGCCCTTCTTATCGC CGGGATTTCCCAGTCATGTTCAGTCGCCGAGATTGAAGAGGCGCTCCAAGCCGGGCTTGCTCCTTTAGGCG AGTATCGTCTCCTTGGGCGGATGTTTCGCCGCGATGAAAATCGCAAAGTAGCGTTGGTTGGTCTCACAGCT GAAACTAGCCATGCGCTTGTACCTAAAGAAATTCCTGGTAAAGGCGGGATCTGGCGGGTTATTTTTAAACC ACCGGACCCGGACAATACGTTTCTTTCTCGTTTGAATGAGTTCCTCGCGGGCGAGGGGATGACGGTGGGG GAACTTAGTCGTGCTCTTGGTCACGAAAATGGGTCATTAGACCCTGAACAGGGTATGATTCCGGAAATGTG GGCGCCGATGCTGGCACAGGCTCTGGAGGCTCTCCAACCGGCTTTACAGTGCCTTAAGTACAAGAAGCTGC GCGTTTTTTCAGGGCGCGAGTCTCCAGAGCCGGGTGAGGAGGAATTCGGCCGTTGGATGTTCCATACCACC CAGATGATCAAAGCGTGGCAGGTGCCGGATGTCGAGAAACGCCGCCGGCTGTTGGAATCACTCCGCGGGC CGGCACTTGACGTTATTCGGGTTCTGAAAATTAACAACCCGTTAATTACGGTAGATGAATGTTTGCAAGCAC TTGAAGAGGTCTTTGGGGTGACTGACAATCCTCGGGAATTGCAAGTAAAATACTTAACGACCTACCATAAG GACGAGGAGAAATTATCAGCCTACGTACTGCGGCTGGAACCGCTGCTGCAGAAGCTCGTCCAGCGGGGGG CTATTGAACGGGACGCTGTTAATCAGGCTCGCCTGGATCAGGTAATCGCTGGGGCGGTACATAAAACTATC CGCCGTGAGCTGAACCTGCCTGAAGACGGGCCGGCGCCAGGCTTTCTTCAACTCCTCGTTTTGATTAAGGA TTACGAGGCAGCTGAAGAGGAGGAAGCATTACTTCAGGCCATTCTTGAAGGGAACTTTACT SEQ ID NO: 56 GGGACAGAACGGCGTCGCGACGAATTAAGTGAAGAAATTAATAATCTTCGTGAAAAGGTTATGAAACAGA GTGAGGAAAACAACAATCTTCAATCCCAAGTCCAGAAACTCACTGAGGAGAATACTACACTCCGTGAGCAA GTTGAACCTACACCTGAAGATGAAGATGACGACATTGAGTTGCGGGGCGCAGCAGCCGCAGCCGCGCCTC CGCCGCCGATCGAGGAGGAATGCCCGGAGGATTTACCGGAAAAATTTGATGGTAATCCGGACATGTTAGC GCCATTCATGGCCCAGTGCCAAATTTTTATGGAAAAGTCTACGCGCGATTTTAGTGTAGATCGCGTACGTGT ATGTTTTGTGACGAGCATGATGACTGGTCGCGCAGCCCGTTGGGCGTCAGCGAAATTGGAGCGGTCGCAC TACCTGATGCATAATTACCCGGCGTTCATGATGGAGATGAAACACGTGTTTGAAGACCCGCAGCGGCGGG AGGTGGCCAAACGCAAGATCCGGCGGTTGCGGCAGGGCATGGGCAGCGTAATTGATTATAGTAATGCGU TCAAATGATTGCGCAGGATCTGGATTGGAATGAACCTGCTCTCATTGATCAATATCATGAAGGGCTTAGTG ACCATATTCAAGAGGAACTCTCTCACCTGGAAGTGGCTAAATCTCTCTCCGCCCTTATTGGCCAATGCATTC ATATTGAGCGCCGTCTTGCACGTGCTGCTGCCGCTCGGAAACCGCGTAGTCCACCACGGGCTTTAGTGCTC CCACATATCGCGTCACACCATCAAGTAGATCCTACTGAGCCAGTGGGGGGTGCACGCATGCGCTTAACCCA AGAAGAAAAGGAACGTCGTCGTAAGCTGAATTTATGCCTGTACTGCGGCACTGGTGGCCATTATGCCGATA ACTGTCCTGCCAAAGCCAGTAAGTCAAGCCCGGCTGGGAAACTTCCAGGTCCTGCCGTCGAGGGCCCTTCT GCTACCGGCCCAGAGATTATCCGCTCCCCGCAAGACGATGCGTCGTCGCCTCATCTCCAGGTAATGCTCCAA ATCCACCTCCCTGGCCGGCACACACTCTTTGTCCGGGCGATGATTGACTCTGGGGCGTCTGGTAATTTTATT GATCACGAGTATGTTGCTCAAAATGGTATCCCTCTCCGGATCAAAGACTGGCCTATTCTGGTTGAAGCCATC GATGGCCGTCCGATCGCGAGCGGTCCTGTGGTTCATGAAACGCATGACCTCATCGTTGATCTGGGTGACCA CCGTGAAGTATTATCCTTTGATGTGACTCAGTCACCGTTTTTTCCAGTTGTTTTGGGCGTCCGTTGGCTTTCG ACTCACGATCCTAACATCACGTGGTCGACACGGTCGATTGTCTTCGATTCGGAATATTGTCGTTATCATTGC CGCATGTATTCACCAATTCCGCCGTCTCTCCCGCCGCCTGCGCCGCAACCTCCTCTGTATTACCCGGTGGAC GGTTACCGTGTTTACCAGCCAGTTCGCTACTACTACGTACAAAACGTGTACACGCCTGTTGATGAACACGTG TACCCAGATCACCGCCTGGTCGACCCTCATATTGAGATGATCCCGGGTGCGCACTCGATCCCATCGGGCCAT GTTTATTCCTTGTCTGAGCCAGAAATGGCCGCCTTACGGGATTTTGTGGCCCGGAATGTCAAAGACGGCCT GATTACCCCGACAATTGCACCAAACGGTGCTCAGGTGTTGCAGGTGAAGCGGGGCTGGAAGTTGCAAGTC AGCTATGATTGTCGTGCGCCAAACAACTTCACTATTCAGAACCAATATCCACGTCTCAGCATCCCTAATCTCG AGGACCAGGCACATCTTGCAACATATACTGAATTTGTACCTCAGATTCCTGGCTATCAGACTTATCCTACGT ATGCTGCCTACCCAACATACCCGGTAGGTTTCGCATGGTACCCAGTAGGCCGGGACGGGCAGGGCCGCTCT TTATATGTTCCTGTCATGATTACATGGAACCCGCATTGGTACCGCCAGCCTCCGGTCCCACAGTACCCACCTC CTCAACCTCCACCACCTCCGCCGCCTCCTCCACCGCCACCTTCTTACTCGACATTA 

1. A capsid comprising a recombinant endogenous Gag polypeptide and a heterologous cargo. 2-25. (canceled) 